For Peer Review Only Non-canonical immunomodulatory activity of complement regulator C4BP limits the development of lupus nephritis Journal: Kidney International Manuscript ID KI-12-18-1865.R2 Article Type: Basic Research Date Submitted by the Author: n/a Complete List of Authors: Luque, Ana; IDIBELL Serrano, Inmaculada; IDIBELL Ripoll, Elia ; IDIBELL Malta, Catarina; IDIBELL Goma, Montse; Hospital Universitari de Bellvitge Blom, Anna; Lund University Grinyó, Joseph; Hospital Universitari de Bellvitge Rodríguez de Córdoba, Santiago; Centro de Investigaciones Biologicas Torras, Juan; Hospital Universitari de Bellvitge Aran, Josep; IDIBELL Subject Area: Chronic Kidney Injury, Immunology, Renal Pathology Keywords: complement, gene expression, glomerulonephritis, inflammation, macrophages, systemic lupus erythematosus The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International For Peer Review Only 1 Non-canonical immunomodulatory activity of complement regulator C4BP limits the development of lupus nephritis Ana Luque1,6, Inmaculada Serrano1,6, Elia Ripoll2, Catarina Malta1, Montserrat Gomà3, Anna M. Blom4, Josep M. Grinyó2, Santiago Rodríguez de Córdoba5, Joan Torras2 and Josep M. Aran1 1 Immune-inflammatory Processes and Gene Therapeutics Group, IDIBELL, 08908 L’Hospitalet de Llobregat, Barcelona, SPAIN. 2 Nephrology Department, Bellvitge University Hospital, Experimental Nephrology Lab., University of Barcelona and IDIBELL, 08908 L’Hospitalet de Llobregat, Barcelona, SPAIN. 3 Pathology Department, Bellvitge University Hospital, IDIBELL, 08908 L’Hospitalet de Llobregat, Barcelona, SPAIN. 4 Lund University, Department of Translational Medicine, Section of Medical Protein Chemistry, 21428 Malmö, SWEDEN. 5 Centro de Investigaciones Biológicas (CSIC) and Ciber de Enfermedades Raras (CIBERER), 28040 Madrid, SPAIN. 6 Equal contributors Correspondence should be addressed to J.M.A. (jaran@idibell.cat): Dr. Josep M. Aran Human Molecular Genetics Group Institut d’Investigació Biomèdica de Bellvitge (IDIBELL) Hospital Duran i Reynals Gran Vía s/n km 2,7 08908 L’Hospitalet de Llobregat Barcelona, SPAIN Phone: +34 (93) 2607428 Fax: +34 (93) 2607414 E-mail: jaran@idibell.cat Running title: C4BP(-) reduces lupus nephritis Keywords: C4BP(-), lupus nephritis, dendritic cells, inflammation, immunomodulation, ectopic lymphoid structures Page 1 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 2 ABSTRACT Lupus nephritis (LN) is a chronic autoimmune-inflammatory condition that can lead to end-stage renal disease because of the breakage of immune self-tolerance occurring in systemic lupus erythematosus (SLE) patients. Presently available immunosuppressive treatments for LN are suboptimal and can induce significant side effects. We have recently characterized a novel immunomodulatory activity on the minor isoform of the classical pathway complement inhibitor, C4BP(-). We show here that C4BP(-) treatment prevented the development of proteinuria and albuminuria, decreased significantly the formation of anti-dsDNA antibodies and, locally, mitigated renal glomerular IgG and C3 deposition and generation of apoptotic cells, with the consequent histological improvement and increased survival in lupus-prone mice. The therapeutic efficacy of C4BP(-) was analogous to that of the broad-acting immunosuppressant cyclophosphamide (CYP). Remarkably, a comparative transcriptional profiling analysis revealed that: 1) the renal gene expression signature resulting from C4BP(-) treatment turned out to be 10 times smaller than that induced by CYP treatment, and 2) C4BP(-) immunomodulation induced significant downregulation of LN relevant transcripts indicating immunopathogenic cell infiltration, including activated T cells (Lat), B cells (Cd19, Ms4a1, Tnfrsf13c), inflammatory phagocytes (Irf7) and neutrophils (Prtn3, S100a8, S100a9). Furthermore, cytokine profiling and immunohistochemistry confirmed that C4BP(-), through systemic and local CXCL13 downregulation, was able to prevent ectopic lymphoid structures neogenesis in aged LN mice. Thus, because of its anti-inflammatory and immunomodulatory activities and high specificity, C4BP(-) could be considered for further clinical development in SLE patients. Page 2 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 3 TRANSLATIONAL STATEMENT Lupus nephritis (LN) is a chronic autoimmune-inflammatory condition for which current treatments with NSAIDs and immunosuppressive drugs, and even with new biologics, remain unsatisfactory and/or induce non-specific adverse events. Therefore, novel therapeutic agents are needed, with increased potency, selectivity and safety profiles. We have recently characterized a novel immunomodulatory activity in the minor isoform of the complement inhibitor C4BP(-). Here we show that this non-canonical activity limits the evolution of LN in lupus-prone mice, preventing the development of CXCL13-driven renal ectopic lymphoid structures. Thus, the anti- inflammatory and tolerogenic functions of C4BP(-) might contribute significantly to restore immune homeostasis and to achieve better outcomes in LN management. Page 3 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 4 INTRODUCTION Systemic lupus erythematosus (SLE) is heterogeneous in nature regarding organ involvement, clinical manifestations and severity, although the most life-threatening trait is progressive nephritis, leading to end-stage renal disease.1 A key early event in lupus nephritis (LN) involves impaired myeloid phagocytes which, unable to clear apoptotic cell debris and/or neutrophil extracellular traps (NETs), initiate a TLR-mediated pro-inflammatory program leading to direct activation of effector T cells and indirect activation of B cells through the production of important mediators such as B-cell activating factor (BAFF).2–4 In turn, stimulation and increased survival of B cells leads to the production of pathogenic autoantibodies against nuclear components (e.g., nucleic acids and histones), local parenchymal immune complex (IC) deposition and activation of the complement system, and additional production of pro- inflammatory chemokines and cytokines such as IFN-I.5 These events induce a self-sustaining feed-forward loop of chronic inflammation and progressive glomerular, tubulointerstitial and endothelial kidney damage. In fact, focal infiltration and progressive organized aggregation of immune cells is the hallmark of autoreactive ectopic lymphoid structures (ELS), also known as tertiary lymphoid organs, developing in both human and murine LN.3,6 Thus, circulating and resident immune cells become important causative agents in the promotion of pro-inflammatory renal injury through ELS expansion, which maximizes the encounters between autoantigens, antigen-presenting cells and lymphocytes, important for the local initiation and maintenance of adaptive immune responses. This knowledge framework offers insight for the development of targeted immunotherapy interventions able to halt tissue damage and chronic inflammation by restoring operational immune tolerance. Accordingly, a new group of biologics, mostly neutralizing antibodies directed against pro-inflammatory molecular targets or cellular leukocyte subsets, such as rituximab, eculizumab, or belimumab, are on pre-clinical or early clinical development aiming to become efficient immunomodulatory agents without the unwanted effects Page 4 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 5 of broad-based immunosuppression displayed by conventional therapy, such as long-term toxicity by cyclophosphamide (CYP) and steroids. However, thus far the therapeutic benefit of biologics for inflammatory renal disease has been limited.7 Synthesized mainly in the liver, C4b-binding protein (C4BP), a main regulator of the complement system, is relatively abundant in the circulation as a major heterooligomer (71, or C4BP(+)), and a minor homooligomer (70, or C4BP(-)) that becomes significantly upregulated under inflammatory conditions (e.g., acute phase response).8,9 Both isoforms are able to inhibit the classical and the lectin pathways of complement activation.10 In addition C4BP(+), through its -chain, forms a high-affinity complex with protein S (PS), which endows it with additional roles in coagulation and in apoptotic cell binding.11,12 Alternatively, we have recently described a novel anti-inflammatory and immunomodulatory activity of human C4BP(-) capable to induce a myeloid-derived suppressor cell (MDSC)-like phenotype through its direct action on monocyte-derived dendritic cells (Mo-DCs), an established model of inflammatory DCs.13 In this study we aimed to assess the therapeutic potential of C4BP(-) in experimental autoimmune LN. We show that C4BP(-), but not C4BP(+), mitigates the development of renal pathology in lupus-prone NZBW F1 and MRL-lpr mice, preventing the neogenesis of autoreactive ELS, which hold a key role locally enhancing renal maladaptive immune response and chronic inflammation. Page 5 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 6 RESULTS C4BP(-) preserves renal function in lupus-prone NZBW F1 mice As we previously reported,13 human C4BP(-) confers a semi-mature, anti-inflammatory phenotype to LPS-matured Mo-DCs from healthy individuals. Thus, we first sought to confirm the immunomodulatory activity of human C4BP(-) in a mouse background. C4BP(-) treatment prevented the upregulation of CD80 and CD86 co-stimulatory molecule expression by cultured bone marrow-derived DCs stimulated with the TLR7 agonist gardiquimod, of relevance in LN pathology.14 (Supplementary Figure S1). To assess the therapeutic potential of C4BP(-) in experimental autoimmune LN we designed an intraperitoneal administration schedule for this human plasma-purified protein into lupus-prone NZBW F1 mice (Figure 1a). Proteinuria is the most prominent and life-threatening sign in LN mice. It reflects renal dysfunction and closely correlates with disease outcome. In PBS vehicle-treated control mice, proteinuria started to develop at week 28 and progressed exponentially to severe proteinuria (> 300 mg/kg) by week 34 (8.5 months of age) up to the end of the study (week 36). In comparison, the onset of proteinuria was delayed until the end of the study by C4BP(-) treatment, and was significantly reduced from that of the control PBS vehicle-treated group (p<0.0001) (Figure 1b). Thus, neither the standard CYP treatment group nor the C4BP(-) treatment group did develop noteworthy proteinuria over time. Nonetheless, a slight upturn in proteinuria, particularly evident as microalbuminuria, appeared by week 36 in the C4BP(-) treatment group, compared with the CYP treatment group (Figure 1c). This apparently decreased therapeutic efficacy of C4BP(-) at later stages of treatment could reflect accelerated clearance due to the development of an immune xenoresponse against the administered human protein in the NZBW F1 immunocompetent mice.15 Certainly, repeated Page 6 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 7 C4BP(-) administration induced the production of anti-C4BP(-)-specific antibodies that were detected in the sera of the treated animals, peaking around week 32 (Supplementary Figure S2). In an additional set of experiments, we confirmed that as little as 50 g/mouse of purified C4BP(-) produced in HEK293 mammalian cells, administered biweekly and subcutaneously, was still able to ameliorate the renal function and increase the survival of nephritic NZBW F1 mice, despite the comparable immunogenicity of both plasma-purified and recombinant C4BP(- ) (Supplementary Figure S3). C4BP(-) attenuates the development of anti-dsDNA antibodies and ameliorates histologic damage in the kidneys of lupus-prone NZBW F1 mice The autoantibody titers directly reflect autoimmunity status. In the vehicle PBS-instilled control NZBW F1 mice, the serum anti-dsDNA total Igs began to progressively increase at 5-6 months of age (week 20-24). Within another 3 months the anti-dsDNA Igs reached its highest titer, which persisted thereafter (Figure 2a). In comparison, mice from the C4BP(-) group had a sustained lower level of autoantibodies throughout the analysis period (weeks 28 to 36) (p<0.05). As expected, the standard CYP treatment group also showed reduced anti-dsDNA autoantibody levels. Even though proteinuria reflects the severity of renal dysfunction, the pathological study directly reveals tissue damage in LN. Common characteristics of severe nephritis in lupus kidneys include proliferative changes in the mesangial and endothelial cells of the glomeruli, capillary basement membrane thickening, severe interstitial infiltrates, tubular atrophy and large protein casts. Figure 2b-d compares representative renal histologic findings in the C4BP(-) and CYP treatment groups and in the PBS vehicle control group. The 9-month old control PBS mice exhibited typical nephritis changes, including significantly enlarged and hypercellular glomeruli Page 7 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 8 (Figure 2c), interstitial inflammation and widespread proteinaceous tubular casts, as expected from their severe proteinuria. Conversely, age-matched mice in both the C4BP(-) and CYP treatment groups presented almost intact glomeruli, tubules and interstitium, with lower pathologic scores (Figure 2b-d). Thus, C4BP(-) treatment attenuated the development of anti- dsDNA antibodies, protecting kidneys from autoantibody induced damage. Renal IC deposits and apoptotic cells are significantly reduced in C4BP(-)-treated lupus- prone NZBW F1 mice IC deposits in the kidneys provide direct evidence for the pathogenicity of the autoantibodies in the target organ and are associated with nephritis in NZBW F1 mice.16 We stained renal cryosections for IgG and C3, two major components in IC. In the vehicle-treated PBS control mice, there was pronounced IgG and C3 deposition in the glomeruli (Figure 3a) which was markedly diminished both in C4BP(-)-treated and in CYP-treated mice (p<0.0001). Therefore, the IC deposits were consistent with the renal histopathological changes. Impaired clearance of apoptotic debris and IgG ICs by phagocytes is considered to be one of the main causes of inflammation associated with LN.17 Indeed, TUNEL analysis revealed widespread presence of both intraglomerular and extraglomerular apoptotic cells in the kidneys of PBS-treated mice. In contrast, both C4BP(-)- and CYP-treated mice had essentially no apoptotic cells in the renal cortex (Figure 3b), correlating with reduced kidney damage. C4BP(-) targets molecular/cellular hallmarks of LN Page 8 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 9 To gain further insight into the local immunomodulatory events induced by C4BP(-) treatment at the molecular and cellular level, we performed a comparative transcriptional profiling of renal tissue from C4BP(-)-treated, CYP-treated, and PBS-treated control NZBW F1 mice. The in-house designed array included 377 genes involved in SLE pathology (Supplementary Table S1). Remarkably, an initial evaluation of the expression profiles of C4BP(-)-treated and CYP-treated mice, both relative to PBS-treated control mice, unveiled a drastically different appearance in their overall shape (Figure 4a). Accordingly, only 24 differentially expressed transcripts (FC > 1.8) were apparent between the C4BP(-)-treated and the PBS-treated kidneys (Supplementary Table S2). Conversely, the same analysis when comparing CYP-treated and PBS-treated kidneys yielded 224 differentially regulated transcripts (Supplementary Table S3). Taking into account the comparable therapeutic efficacies of C4BP(-) and CYP treatments in LN mice, as perceived from the previously analyzed parameters, this outcome suggests more specific immunomodulation induced by C4BP(-) over the immunosuppressant CYP in LN NZBW F1 mice. An additional gene-interaction based bioinformatics analysis of the analyzed genes revealed that relevant biological functions modulated by C4BP(-)-treatment involved reduced renal leukocyte migration, particularly of myeloid cells, and consequently, a decreased quantity and activation of infiltrated mononuclear leukocytes, predominantly B lymphocytes (Supplementary Table S4). Conversely, a great number of additional relevant pathways, such as the complement pathway, were appreciably regulated only by CYP treatment (data not shown) suggesting increased toxicity of the immunosuppressant CYP over C4BP(-). Next, we validated the above results obtained with the microfluidics cards by analyzing renal tissue from individual mice within each group by RT-qPCR, and interrogating the transcripts that showed differential regulation by C4BP(-) at FC > 2.0 in the array. We confirmed significant differential C4BP(-)-mediated downregulation of 8 transcripts: Irf7, Lat, Page 9 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 10 Cd19, Ms4a1 (Cd20), Tnfrsf13c (BAFFR), Prtn3, S100a8 and S100a9 (Figure 4b). All but S100a8 and S100a9, encoding the S100a8/S100a9 complex calprotectin involved in inflammasome activation and myelopoiesis induction, were found also significantly downregulated by CYP treatment. Strikingly, while Irf7 is a key transcription factor involved in the type I IFN pathway activated by innate sensors, such as TLR7/8, as a consequence of impaired lysosomal maturation of SLE phagocytes,2 all other transcripts are cellular markers specific for activated T lymphocytes (Lat), B lymphocytes (Cd19, Ms4a1, Tnfrsf13c), and neutrophils (Prtn3, S100a8, S100a9). This again indicates significant leukocyte infiltration, suggesting a lack of immune tolerance as the underlying cause of LN pathology in the kidneys of PBS-treated control NZBW F1 mice which, however, could be mitigated by both C4BP(-) and standard CYP treatment. Further, assessment of network crosstalk between the C4BP(-) gene set including the above 8 significantly regulated genes and the KEGG pathways using PathwAX reflected significant (FDR < 0.05) depletion of immune hematopoietic cells and, particularly, of the B cell signaling and the pro-inflammatory NF-B pathways in C4BP(-)-treated renal tissue compared with the PBS-treated, nephritic tissue (Table 1). C4BP(-) modulates the cytokine signature of lupus-prone NZBW F1 mice To complement the transcriptional profiling results obtained, we examined the presence of inflammatory cytokines in the sera of the treated NZBW F1 mice at the end of the study. Remarkably, a cytokine array including 40 mouse cytokines, chemokines and acute phase proteins evidenced CXCL13 as the unique cytokine differentially abundant, present in PBS- treated mice but absent in C4BP(-)-treated or CYP-treated mice (Figure 5a). This homeostatic B cell-attracting chemokine, produced by macrophages and dendritic cells, is extremely relevant Page 10 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 11 in SLE renal pathology. We confirmed the array findings assessing the CXCL13 levels in the sera of individual mice through a specific ELISA assay (Figure 5b). An identical cytokine analysis, using renal tissue extracts instead of sera, endorsed differential expression of CXCL13 and, additionally, revealed local presence of the C5/C5a complement components, and of the IL1 family members IL1 receptor antagonist (IL1ra; IL- 1F3) and IL1 (Figure 5c and 5d). Thus, local increase of these inflammatory factors, while apparent in PBS-treated kidneys, could be prevented by C4BP(-) or CYP treatment. C4BP(-) treatment prevents leukocyte infiltration and the development of ELS in the renal parenchyma of lupus-prone NZBW F1 mice LN, a chronic inflammatory condition, features significant leukocyte infiltration over time, as suggested by our transcriptional profiling data. Indeed, a thorough immunohistochemical examination regarding the presence of inflammatory leukocyte markers in the renal cortex of 36 weeks old PBS-treated NZBW F1 mice evidenced positive staining, mainly perivascular and, occasionally, periglomerular, not only for activated T cells (Lat) and B cells (CD19), but also for different myeloid cell populations: neutrophils (Gr1), interstitial monocytes/macrophages (F4/80), and dendritic cells (CD11c). In contrast, the above markers were barely present (F4/80) or virtually absent in both C4BP(-)-treated or CYP-treated mice (Figure 6). The chemokine CXCL13 holds a key role recruiting and organizing nodular aggregates of B cells, activated T cells and DCs surrounded by neo-lymphatic vessels termed ELS.18 Indeed, we confirmed the presence of multiple ELS in the cortex of PBS-treated NZBW F1 nephritic mice by hematoxilin and eosin staining and by T cell (Lat) and B cell (Cd19) immunostaining (Figure 7). In contrast, C4BP(-) immunomodulatory activity prevented the development of Page 11 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 12 local inflammatory ELS in these mice, which correlated with reduced autoantibody anti-dsDNA formation and renal function improvement. The complement inhibitory activity of C4BP does not affect LN pathology in lupus-prone NZBW F1 mice To assess whether C4BP-mediated complement inhibition could be responsible for the improvement of LN we performed a comparative study administering equal amounts of either the minor C4BP(-) isoform, sustaining both complement inhibitory and immunomodulatory activities, or the major isoform C4BP(+), holding only the complement inhibitory but not the immunomodulatory activity,13 into NZBW F1 mice. C4BP(-)-treated mice showed reduced proteinuria, decreased anti ds-DNA and blood urea nitrogen (BUN) levels, improved renal histology with minimal immune cell infiltration and a higher survival rate when compared with C4BP(+)-treated mice (Supplementary Figure S4). Furthermore, we analyzed the activity of the classical pathway of complement in fresh serum extracted from NZBW F1 mice previously (week 21), and at several points during C4BP treatment (weeks 25, 29 and 33). The complement activity was nearly within the normal range in young mice, before starting C4BP treatment, but was concomitantly reduced with the progression of LN pathology in both the PBS- and C4BP(+)-treated mice (Supplementary Figure S5). A severe drop in hemolytic complement in advance of clinical renal disease has been reported in these mice, which suggested consumption by ICs.19 In contrast, C4BP(-) treatment significantly precluded hypocomplementemia at the early stages of treatment. Nevertheless, complement exhaustion could not be prevented at the later stages of treatment, possibly due to lack of C4BP(-) immunomodulatory efficacy because of the development of the anti-human C4BP(-) immune xenoresponse, as previously indicated. Page 12 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 13 Therapeutic efficacy of C4BP(-) in lupus-prone MRL-lpr mice We substantiated the above results and the overall therapeutic efficacy of C4BP(-) immunomodulation using MRL-lpr mice as an alternative spontaneous LN model. Thus, a weekly treatment schedule using 100 g C4BP(-)/mouse starting at 10 weeks of age not only mitigated the development of proteinuria, increased survival, preserved renal histology preventing ectopic lymphoid tissue formation and increased the survival rate, but also ameliorated dermatitis and vasculitis, other SLE pathological traits developing in the MRL-lpr model (Figure 8). Page 13 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 14 DISCUSSION In this report we demonstrate the benefit of human C4BP(-), an acute phase protein with a well-known complement inhibitory function and with a previously unrecognized and recently characterized anti-inflammatory and tolerogenic activity towards inflammatory DCs, in two spontaneous models of autoimmune LN: NZBW F1 and MRL-lpr mice. A prior study has reported the potential of human C4BP(-) inhibiting the development of autoimmune arthritis in mice, which was exclusively attributed to its complement inhibitory activity.20 However, the fast systemic clearance of human C4BP(-) when administered to mice 20 suggests that an alternative mode of action of C4BP(-) might contribute significantly to the substantial therapeutic effect noticed in the arthritis models employed. In fact, we show here that the major C4BP(+) isoform, holding the same complement inhibitory activity than C4BP(-) but lacking immunomodulatory activity, 13 does not affect the development of LN from NZBW F1 mice. Furthermore, C4BP(-), but not C4BP(+), was able to prevent in these mice the progress to hypocomplementemia, an important marker for the presence of IC-mediated disease. It has been shown that complement titers fall significantly with renal disease progression, concomitantly with the appearance of anti-DNA autoantibodies in serum and of C3 Igs in renal glomeruli.19 Nonetheless, we acknowledge C4BP(-) action limiting both C3 deposition in the glomeruli and the presence of C5/C5a in the renal cortex of C4BP(-)-treated NZBW F1 mice. In fact, inhibition of complement activity at different points, such as through mAb-mediated C5 blockade,21 using C3a22 or C5a23 receptor antagonists, or by CR2-targeted complement inhibitors: CR2-DAF,24 CR2-Crry,25 or CR2-FH26 has proven useful in the amelioration of LN. Yet the deposition of ICs or complement does not seem sufficient for renal pathology.27–29 In our study, while CYP, a broad-acting immunosuppressant and standard of care treatment for the most severe manifestations of SLE,30 induced transcriptional downregulation of several Page 14 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 15 complement effectors in renal tissue from NZBW F1 mice, no such modulation was observed upon C4BP(-) treatment. Nevertheless, the therapeutic efficiency of C4BP(-) was comparable to that exhibited by CYP. Moreover, scaling down 10-fold the subcutaneous dosage of C4BP(-) to 50 g/mouse and setting up a bi-weekly administration schedule, was still able to significantly ameliorate disease severity in LN mice. Mouse C4BP lacks both the -chain,31 and two complement control protein domains (CCPs) in the -chain homologous to CCP5 and CCP6 from the human C4BP -chain.32 We have previously described that CCP6 is necessary for the immunomodulatory activity of human C4BP(-).13 Hence, the mouse constitutes a suitable animal model to assess the immunomodulatory activity of human C4BP(-) without interference from endogenous mouse C4BP. Certainly, C4BP knock-out did not develop any autoimmune phenotype, and did not modify disease severity in MRL-lpr LN mice,33 stressing the relevance of human C4BP(-) immunomodulation in NZBW F1 LN pathology. Both C4BP(-),13 and the major alternative pathway complement inhibitor FH,34 through as yet unknown receptor(s), directly counteract cellular immune-inflammatory activation by inducing an anti-inflammatory and tolerogenic state on monocyte-derived DCs analogous to that recently described for monocytic MDSCs, which confer suppressive activity toward multiple myeloid and lymphoid cell subsets.35,36 Remarkably, myeloid cells seem to take a central pathogenic role in SLE owing to inefficient clearance of apoptotic cell debris and autoantigen- autoantibody ICs by phagocytes because of defects in lysosomal maturation.2 Thus, FcRI- mediated activation of DC and macrophage innate sensors, through accumulated IgG-ICs in their surfaces, seems to occur prior to significant B cell expansion, IFN- and BAFF secretion, and LN progression.17 For example, RNA sensing by conventional DCs through TLR7 has been reported to be critical to the development of LN.37 C4BP(-) is able to prevent TLR4- and TLR7-mediated activation of monocyte-derived DCs (13, and data not shown). Accordingly, Page 15 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 16 immunomodulatory C4BP(-) administration in our NZBW F1 LN model seemed to preclude glomerular and interstitial apoptotic cell accumulation and/or apoptotic debris coating of myeloid cells, either preventing cell dying or increasing their renal clearance. Strikingly, at the molecular level, a comparative analysis of the transcriptional fingerprints of C4BP(-), CYP, and the vehicle PBS induced in renal tissue from 36-week old NZBW F1 mice uncovered the high specificity of C4BP(-) treatment respect to the standard CYP regimen. Thus, setting the threshold FC to + 1.8, only 24 transcripts (6.4 % of the SLE genes arrayed) were regulated by C4BP(-) compared with 224 transcripts (59.4 % of the SLE genes arrayed) by CYP, nevertheless both conferring analogous functional improvements in the LN NZBW F1 kidneys. This points out to increased safety due to a much more focused and specific action of C4BP(-)-mediated immunomodulation as compared with the known toxicity profile concomitant to CYP global immunosuppression. The resulting C4BP(-) transcriptional profile also revealed exclusive downregulation of typical immune cell markers, with a significant reduction of B cell (Cd19, Cd20, BAFFR), T cell (Lat) and neutrophil (Prtn3, S100a8, S100a9) transcripts identifying inflammatory cell infiltrates in the renal parenchyma, relative to untreated nephritic NZBW F1 kidneys. Of note, the alarmin calprotectin (S100a8/S100a9), exclusively downregulated by C4BP(-) treatment, induces inflammasome activation and IL-1-dependent monocytosis and neutrophilia.38 Concordantly, it has been recently shown that human C4BP inhibits pancreatic islet amyloid polypeptide (IAPP)- induced inflammasome activation.39 Indeed, the reduction/absence of both lymphoid and myeloid cells in C4BP(-)-treated kidneys was backed by immunohistochemical evaluation and confirms that C4BP(-) immunomodulation targets cellular effectors responsible for the immune dysregulation driving SLE.40 Moreover, experimental and bioinformatics analyses stressed the inhibition of the pro-inflammatory NF-kB and interferon (Irf7) pathways through C4BP(-) Page 16 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 17 action, leading to suppression of innate immune responses. Both pathways have deemed essential for autoantibody production and development of nephritis in murine lupus.41 Chronic, unresolved inflammation in LN paves the way for ELS development, a hallmark of tissue autoimmunity, which intends to address high local concentration of autoantigens supporting the formation of a network of follicular DCs (FDCs) for antigen presentation, surrounded by proliferating/activated B- and T-cell rich areas, much like secondary lymphoid organs. It has been shown both in the NZBW F1 model and in LN patients that the chemokine CXCL13 plays an important role in the initiation and development of LN,42,43 and abnormally increased serum CXCL13 levels could induce extensive chemotaxis of CXCR5-expressing B cells, activated T cells and DCs into renal LN tissues stimulating inflammatory ELS formation.18,44 Conversely, it has been shown that direct CXCL13 blockade disrupts ELS formation45 and attenuates LN in MRL-lpr mice.46 Remarkably, C4BP(-) treatment leads also to significant CXCL13 downregulation both systemically and locally in NZBW F1 kidneys. Consequently, the presence of cortical ELS in our C4BP(-)-treated aged mice was virtually absent, consistent with substantially reduced inflammatory immune cell infiltrates compared with untreated nephritic aged mice. FDCs have been considered the main producers of CXCL13, although newly recruited monocytes, macrophages and myeloid DCs have also been shown to secrete CXCL13 in response to activation by TLR2/4 ligands.47–51 Thus, C4BP(-) action through the inflammatory myeloid cell-CXCL13-ELS axis would prevent ELS neogenesis in chronically inflamed tissues, which has been correlated with allograft rejection and autoimmune disease progression.52 In summary, additional studies will be required to further decipher the molecular mechanism of C4BP(-) immunomodulation leading to improvement of LN pathology. Nevertheless, the relevant efficacy and specificity of C4BP(-) in both NZBW F1 and MRL-lpr Page 17 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 18 LN mice anticipates promising possibilities to explore novel therapeutic options for LN SLE patients. Page 18 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 19 CONCISE METHODS Proteins and drugs Peripheral blood from healthy individuals was supplied by the local blood bank. The C4BP(+) heterooligomer was purified from pooled plasma by BaCl2 precipitation, as previously described.53 C4BP(-) was obtained from pooled plasma supernatants after BaCl2 precipitation, and purified according to a protocol set up from Bioingenium (Barcelona, Spain). Their purity was higher than 85%, as assessed by SDS-PAGE and Coomassie Blue staining (Supplementary Figure S6). CYP (Genoxal®, Baxter Oncology GmbH, Halle/Westfallem, Germany), was resuspended in saline. Further information is given in Supplementary Methods. Mice, study design and follow up We used NZBW F1 and MRL-lpr female mice 54 (Jackson Laboratory, Bar Harbor, ME, USA) (6 - 8 animals/group), aged 6 months (NZBW F1 mice; 35-40 g/each) or 2.5 months (MRL-lpr mice; 25-30 g/each), for the studies. C4BP isoforms (C4BP(-), rC4BP(-), and C4BP(+)) were administered intraperitoneally (ip) or subcutaneously (sc), as indicated, for 3 months (between 6 and 9 months of age in the case of NZBW F1 mice, and between 2.5 and 5.5 months of age in the case of MRL-lpr mice), at the specified doses and schedules. CYP was also administered ip at 2.5 mg/mouse every 10 days, in the same timeframes, as previously described.55,56 Finally, a control group underwent vehicle PBS administration following the same administration route and schedule as the C4BP(-) group in the different studies (Figures 1a and 8a; Supplementary Figures S3a and S4a). Detailed procedures are given in Supplementary Methods. Renal function analysis: proteinuria, albuminuria and BUN Page 19 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 20 Twenty-four hour urinary protein was determined by the pyrogallol red-molibdate protein-dye binding method (Olympum Autoanalyzer AU400, Hamburg, Germany) while 24 h urinary albumin was determined using a commercially available ELISA KIT (Active motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Levels of BUN in the sera from the NZBW F1 mice were measured using a commercially available enzymatic kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s recommendations. Assessment of anti-dsDNA antibodies Levels of anti-dsDNA antibodies (IgG + IgA + IgM) were measured using a commercially available ELISA kit (Alpha Diagnostic International, San Antonio, TX, USA) according to the manufacturer’s recommendations. Renal histopathology Coronal kidney slices (1–2 mm thick) were fixed in 4% paraformaldehyde and embedded in paraffin. For light microscopy, 5 m thick tissue sections were stained with hematoxylin and eosin (H&E) and analyzed in a Nikon Eclipse 80i microscope (Nikon Instruments, Amstelveen, Netherlands). Glomerular cross-sectional area (µm2) was calculated based on average area of 150 glomeruli in each group. Details are given in Supplementary Methods. Immunofluorescent determination of renal IC and complement deposition For analysis of IgG and C3 deposition, fluorescent staining of kidney cryo-sections was performed. Sections were directly stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Sigma-Aldrich), and FITC-conjugated C3 (Nordic Immunology, Tilburg, Page 20 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 21 The Netherlands). At least 10 glomeruli per section were visualized. For detailed analyses, see Supplementary Methods. Complement activity assay Activity of the classical complement pathway in fresh sera was determined by C3b deposition on K562 cells opsonized with rabbit polyclonal antibodies (Agrisera, Vännäs, Sweden), as previously described,20 and quantified using FITC-conjugated goat IgG fraction to mouse complement C3 antibody (MP Biomedicals, Solon, OH, USA) and flow cytometry. Heat- inactivated serum samples (56 ºC, 30 min) were used as negative controls. A further description is given in Supplementary Methods. Immunohistochemistry The following primary antibodies were used: polyclonal rabbit anti-mouse CD11c (1:200, Biorbyt, BioNova, Cambridge, UK), polyclonal rabbit anti-mouse Lat (1:50, ThermoFisher, Waltham, MA, USA), monoclonal rat anti-mouse Gr1 (1:50, R&D Systems, Minneapolis, MN, USA), monoclonal rat anti-mouse F4/80 (1:50, ThermoFischer), monoclonal rat anti-mouse CD19 (1:200, ThermoFischer). F4/80 marker detection required antigen retrieval using proteinase K. After PBS washing, slices were incubated with the appropriate secondary antibody: biotinylated goat anti-rabbit IgG (1:200, Vector laboratories, Peterborough, UK), or ImmPRESS™ HRP anti-rat IgG (mouse adsorbed) polymer detection kit (Vector laboratories) for 1 h at RT. Regarding CD11c and Lat markers, standard Vectastain (ABC) avidin-biotin peroxidase complex (Vector laboratories) was applied. For further details about tissue processing and staining, see Supplementary Methods. TUNEL Assay Page 21 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 22 Apoptosis detection was performed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) using the “TACS TdT in situ DAB” kit (R&D Systems) according to the manufacturer's instructions. Details are given in Supplementary Methods. Differential gene expression analysis Total RNA from mouse kidneys was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Gene expression profiling was performed by interrogating reverse transcribed pooled cDNA (2-3 animals/group) using custom TaqMan Low Density Arrays (TLDA Cards) (Applied Biosystems, Carlsbad, CA, USA) (Supplementary Table S1) run on the 7900HT system for quantitative real-time PCR analysis according to the manufacturer’s instructions. Differentially expressed genes were analyzed using the Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, Qiagen) to identify biological and molecular networks differentially regulated in the LN model. Selected gene transcripts were further validated in individual mouse kidney samples by RT-qPCR using TaqMan Gene Expression Assays (Applied BioSystems). Pathway annotation of the RT-qPCR-validated gene set was performed through PathwAX.57 Detailed procedures are given in Supplementary Methods. Mouse cytokine array Serum and renal lysates were analyzed using the Proteome Profiler Array “Mouse Cytokine Array Panel A” kit (R&D Systems), according to the manufacturer’s instructions. Further details are given in Supplementary Methods. CXCL13 ELISA Page 22 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 23 CXCL13 cytokine levels from both sera and tissue lysates were quantified using the “Legend Max Mouse CXCL13 (BLC) ELISA” kit (BioLegend, San Diego, CA, USA) according to the manufacturers’ instructions. Statistical analysis Statistical analyses and scientific graphing were performed using the GraphPad Prism 6 software (GraphPad software, Inc, La Jolla, CA, USA). Two-way ANOVA, corrected for multiple comparisons using the Holm-Sidak method, was applied to analyze proteinuria, albuminuria, BUN, anti-dsDNA antibodies, complement activity and dermatitis scores throughout the follow up studies. One-way ANOVA, corrected for multiple comparisons using the Dunnett’s method, was employed to assess histological data and CXCL13 cytokine levels. Meier plots along with the log-rank (Mantel-Cox) test were performed to assess the differences in the survival distributions. Vehicle PBS-treated mice were assigned as the reference group, unless otherwise indicated. Relative gene expression levels between the C4BP(-)- and CYP- treatment groups relative to the vehicle PBS-treated group in the validation step were analyzed using the one sample t-test. The Friedman test, corrected using the Dunn’s post-hoc analysis method, was applied to compare flow cytometry (MFI) data. Unless otherwise stated, data are expressed as mean values + SD. In all cases, a P value < 0.05 was considered significant. Page 23 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 24 SUPPLEMENTARY MATERIAL Figure S1. Figure S2. Figure S3. Figure S4. Figure S5 Figure S6 Table S1. List of relevant SLE genes included in the customized TaqMan Low Density Array. Table S2. List of genes induced by C4BP(-) treatment (FC > 1.8). Table S3. List of genes induced by CYP treatment (FC > 1.8). Table S4. Relevant enriched biological functions (IPA®) associated with differentially expressed renal genes from C4BP(-)-treated NZBW F1 mice Supplementary Methods. Supplementary material is linked to the online version of the paper at www.kidney- international.org. Page 24 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 25 DISCLOSURE JMA is co-inventor on pending or issued patents involving compounds and methods for immunomodulation. The authors report no other conflicts of interest in this work. Page 25 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 26 ACKNOWLEDGEMENTS We thank Nuria Lluch and Jordi Ortiz (Spherium Biomed S.L.) for fruitful discussions and contribution to the research. This work was supported by the Ministerio de Ciencia, Innovación y Universidades (Madrid, Spain) (grants FIS-ISCIII PI16/00377 and PI13/00969, cofunded by FEDER funds/European Regional Development Fund (ERDF)-a way to build Europe-), the Generalitat de Catalunya (grant 2017SGR291, and CERCA Program), and Spherium Biomed S.L. Drs. Aran and Rodriguez de Córdoba are members of the Red de Excelencia “Complemento en salud y enfermedad” (SAF2016-81876-REDT). Dr. Rodriguez de Córdoba is supported by the Spanish “Ministerio de Economía y Competitividad/FEDER” [SAF2015-66287-R] and the Autonomous Region of Madrid [S2017/BMD-3673]. Dr. Aran is sponsored by the “Researchers Consolidation Program” from the SNS-Dpt. Salut Generalitat de Catalunya (Exp. CES06/012). Page 26 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 27 REFERENCES 1. Maroz N, Segal MS. Lupus nephritis and end-stage kidney disease. Am J Med Sci. 2013;346:319-323. 2. Monteith AJ, Kang S, Scott E, et al. Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus. Proc Natl Acad Sci U S A. 2016;113:E2142-51. 3. 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J Immunol. 2016;197:2167-2176. Page 30 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 31 42. Schiffer L, Bethunaickan R, Ramanujam M, et al. Activated renal macrophages are markers of disease onset and disease remission in lupus nephritis. J Immunol. 2008;180:1938-1947. 43. Sang A, Zheng Y-Y, Morel L. Contributions of B cells to lupus pathogenesis. Mol Immunol. 2014;62:329-338. 44. He DN, Chen WL, Long KX, et al. Association of serum CXCL13 with intrarenal ectopic lymphoid tissue formation in lupus nephritis. J Immunol Res. 2016;2016:4832543. 45. Yamamoto K, Nishiumi S, Yang L, et al. Anti-CXCL13 antibody can inhibit the formation of gastric lymphoid follicles induced by Helicobacter infection. Mucosal Immunol. 2014;7:1244-1254. 46. Wu X, Guo J, Ding R, et al. CXCL13 blockade attenuates lupus nephritis of MRL/lpr mice. Acta Histochem. 2015;117:732-737. 47. Carlsen HS, Baekkevold ES, Morton HC, et al. Monocyte-like and mature macrophages produce CXCL13 (B cell-attracting chemokine 1) in inflammatory lesions with lymphoid neogenesis. Blood. 2004;104:3021-3027. 48. Vermi W, Facchetti F, Riboldi E, et al. Role of dendritic cell-derived CXCL13 in the pathogenesis of Bartonella henselae B-rich granuloma. Blood. 2006;107:454-462. 49. Rupprecht TA, Kirschning CJ, Popp B, et al. Borrelia garinii induces CXCL13 production in human monocytes through Toll-like receptor 2. Infect Immun. 2007;75:4351-4356. 50. Moreth K, Brodbeck R, Babelova A, et al. The proteoglycan biglycan regulates expression of the B cell chemoattractant CXCL13 and aggravates murine lupus nephritis. J Clin Invest. 2010;120:4251-4272. 51. Cohen SB, Smith NL, McDougal C, et al. Beta-catenin signaling drives differentiation and proinflammatory function of IRF8-dependent dendritic cells. J Immunol. 2015;194:210- 222. Page 31 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 32 52. Neyt K, Perros F, GeurtsvanKessel CH, et al. Tertiary lymphoid organs in infection and autoimmunity. Trends Immunol. 2012;33:297-305. 53. Mohlin FC, Blom AM. Purification and functional characterization of C4b-binding protein (C4BP). Methods Mol Biol. 2014;1100:169-176. 54. Rottman JB, Willis CR. Mouse models of systemic lupus erythematosus reveal a complex pathogenesis. Vet Pathol. 2010;47:664-676. 55. Alperovich G, Rama I, Lloberas N, et al. New immunosuppresor strategies in the treatment of murine lupus nephritis. Lupus. 2007;16:18-24. 56. Ripoll È, Merino A, Goma M, et al. CD40 gene silencing reduces the progression of experimental lupus nephritis modulating local milieu and systemic mechanisms. PLoS One. 2013;8:e65068. 57. Ogris C, Helleday T, Sonnhammer ELL. PathwAX: a web server for network crosstalk based pathway annotation. Nucleic Acids Res. 2016;44:W105-W109. Page 32 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 33 FIGURE CAPTIONS Figure 1.- Renal function determination in lupus-prone NZBW F1 mice. Schematic administration schedule and dosage for intraperitoneal (ip) injection of C4BP(-) and vehicle (PBS) (blue arrows), and cyclophosphamide (CYP) (red arrows) in lupus-prone NZBW F1 mice (a). Proteinuria and albuminuria were monitored monthly over the course of the assay and all animals were sacrificed at week 36 (w36). Total 24-h urinary protein was determined by Pyrogallol Red-molybdate protein dye-binding assay (b), and 24-h urinary albumin was determined by ELISA (c) as described in Methods. Data are normalized by mouse weight and expressed as mean values + SD (n= 4-8 mice/group); ***p < 0.001; ****p < 0.000,1 compared with control, PBS-treated mice. Figure 2.- Anti-dsDNA autoantibody production and renal histology assessment in lupus- prone NZBW F1 mice. (a) Serum levels of anti-dsDNA antibodies correlating with disease activity were measured by ELISA from week 20 (pretreatment) to week 36. Data are expressed as mean + SD (n= 7 mice/group); *p < 0.05; **p <0.01; ****p <0.0001, compared with vehicle PBS-treated mice. (b) Representative sections of renal cortex from 36-week old NZBW F1 mice treated with CYP (left), C4BP(-) (center) and vehicle PBS (right) mice stained with hematoxylin and eosin. Images are shown at low (upper panels; scale bars: 200 m) and high (lower panels; scale bars: 50 m) magnification. The data obtained were used to comparatively evaluate the glomeruli area (c) and to semi-quantitatively grade histopathology traits indicating active LN (mesangial expansion, endocapillary proliferation, glomerular deposits, extracapillary proliferation and interstitial infiltrates), as well as chronic lesions (tubular atrophy and interstitial fibrosis) (d). Page 33 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 34 Figure 3.- Immunohistochemical analysis of IgG and C3 deposits and evaluation of apoptosis in the renal cortex of lupus-prone NZBW F1 mice. For analysis of local IgG and C3 deposition, fluorescent staining of renal cryo-sections was performed (a). Representative fluorescent images from IgG- and C3-stained vehicle PBS- and C4BP(-)-treated NZBW F1 mice are shown (scale bar: 50 m) (upper panels). Fluorescence was expressed as mean fluorescence intensity (MFI) + SD (n= 7 mice/group); ****p < 0.0001, compared with vehicle PBS mice (lower panels). Apoptosis in the renal cortex was detected by TUNEL assay (b). Representative images from renal cortex of CYP-, C4BP(-)-, and PBS vehicle-treated NZBW F1 mice are shown at low (scale bar: 200 m) and high (scale bar: 50 m) magnification. Scoring of cortical apoptotic cells as cell number per field (left graph) and glomerular apoptotic cells as cell number per glomerular cut section (gcs) (right graph). Data are expressed as mean + SD (n= 3 mice/group); ****p <0.0001, compared with vehicle PBS-treated mice. Figure 4.- Comparative transcriptional profiling of renal tissue from lupus-prone NZBW F1 mice. Overall comparative transcriptional profiles of 36-week renal tissue from CYP- and C4BP(-)- treated mice relative to the 36-week nephritic transcriptional profile displayed by renal tissue from vehicle PBS-treated mice (a). Total renal RNA was interrogated against a panel of 377 murine genes relevant in LN pathology. Gene expression data are given as Log2FC (FC, fold change). The discontinuous horizontal lines indicate a fold change threshold of + 1.8 for upregulated and downregulated genes. TaqMan RT-qPCR individual validation of all genes showing a FC induction > 2 in the C4BP(-) transcriptional profile (b). Relative expression data for the specified genes was obtained from renal RNA of individual mice belonging to each Page 34 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 35 treatment group (n= 3-5 mice/group). Log2FC values are expressed as mean + SD; *p <0.05, **p <0.01 compared with vehicle PBS-treated mice. Figure 5.- Circulating and renal cytokine profile from lupus-prone NZBW F1 mice. Sera (a) and renal tissue extracts (c) were incubated with the R&D Systems “Proteome Profiler Mouse Cytokine Array Kit”, according to the manufacturer’s guidelines. The density of each dot was quantified with Quantity One® software and displayed as normalized mean pixel density for each relevant cytokine and treatment. Additional validation of the chemokine CXCL13 was performed in individual mouse samples (n= 4 mice/group) by a specific ELISA (b and d). Data are expressed as mean values + SD; ****p <0.0001, compared with vehicle PBS-treated mice. Figure 6.- Immunohistochemistry of renal cortex from lupus-prone NZBW F1 mice. Assessment of immune cell infiltrates in renal cortex sections from CYP-, C4BP (-)-, and vehicle PBS-treated aged NZBW F1 mice at the end of the study (36 weeks old). Local intense perivascular, periglomerular and/or glomerular staining was observed in vehicle PBS-treated kidneys surveyed for activated T cells (Lat), B cells (CD19), and myeloid cells such as neutrophils (Gr1), DCs (CD11c), and monocytes/macrophages (F4/80), the latter presenting a strong and diffuse periglomerular staining (lower panels). Conversely, CYP-treated (upper panels) and C4BP(-)-treated (middle panels) kidneys were found scarcely stained for the above- referred inflammatory markers. Scale bars: 50 m. Results from each panel are representative of staining performed on tissue from 3 animals in each group. Figure 7.- C4BP(-) treatment prevents the development of ELS in lupus-prone NZBW F1 mice. Page 35 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 36 Upper panels, low resolution hematoxilin-eosin stained representative renal cortex sections from C4BP(-)-treated (left) and vehicle PBS-treated (right) aged (36 week old) NZBW F1 mice. ELS are clearly visible in the PBS-treated mice (black arrows), but absent in C4BP(-)-treated mice. Scale bars: 200 m (representative images from 4 mice/group). Lower panels, consecutive renal sections of C4BP(-)-treated and PBS-treated mice stained with hematoxilin-eosin (left), and immunostained with Lat (activated T cell marker) (center) and CD19 (B cell marker) (right). Note the co-localization of T and B cells within a well-developed ELS surrounding an arteriole (a) and next to a vein (v) in the higher magnification images from PBS-treated renal cortex. ELS are virtually nonexistent in C4BP(-)-treated renal cortex. Scale bars: 100 m. Figure 8.- C4BP(-) attenuates autoimmune lupus manifestations in MRL/lpr mice. (a) Schematic administration schedule and dosage for intraperitoneal (ip) injection of C4BP(-) and vehicle (PBS) (blue arrows), and cyclophosphamide (CYP) (red arrows) in MRL/lpr mice. (b) Proteinuria was monitored bimonthly (unless otherwise stated) over the course of the assay from week 9 until week 22. Total 24-h urinary protein was determined by Pyrogallol Red- molybdate protein dye-binding assay. Data are normalized by mouse weight and expressed as cumulative mean values (PBS, n= 8; C4BP(-) and CYP, n= 6); *p < 0.05; **p < 0.01 compared with control, PBS-treated mice. (c) Kaplan-Meier survival curves from lupus-prone MRL/lpr mice. Cumulative survival curves showed 100% survival (8/8) in the CYP-treated and C4BP(-)- treated mice, and 62.5% survival (5/8) in PBS-treated mice at the end of the study (day 147). Red arrows identify the start (day 70) and the end (day 147) of the treatment period. n=6-8 mice/group; *p < 0.05, compared with vehicle PBS-treated mice; long-rank test. (d) Representative sections of renal cortex from 22-week old MRL/lpr mice treated with CYP (left), C4BP(-) (center) and vehicle PBS (right) mice stained with hematoxylin and eosin. Images are shown at 200 m magnification. The data obtained were used to semi-quantitatively grade Page 36 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 37 histopathology traits indicating active LN (mesangial expansion, endocapillary proliferation, glomerular deposits, extracapillary proliferation and interstitial infiltrates), as well as chronic lesions (tubular atrophy and interstitial fibrosis) (n= 3 mice/group) (e) Clinical features of dermatitis in MRL-lpr mice treated with C4BP(-) (left panels) or PBS (right panels). Major lesions were observed around the facial and scapular regions, and vasculitis was evident in the ears of PBS-treated mice. Skin lesion severity score at weeks 19 and 21 for each of the study groups is shown on the right (PBS, n= 5; C4BP(-) and CYP, n= 6); *p < 0.05; **p < 0.01 compared with PBS-treated mice. Page 37 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (October 2019) KI-12-18-1865.R1 38 Table 1.- Pathway annotation of NZBW F1 renal genes downregulated by C4BP(-) treatment based on network crosstalk (PathwAX). Pathway class # Relevant pathway FWER Network connectivity of C4BP(β-)-induced genes (Links) Ms4a1 Prtn3 Tnfrsf13c Lat Cd19 S100a8 S100a9 Irf7 Human diseases 14 Systemic lupus erythematosus 2.20x10-3 Organismal systems 2 Hematopoietic cell lineage 1.53x10-14 3 B cell receptor signaling pathway 1.68x10-12 15 Antigen processing and presentation 2.46x10-3 18 T cell receptor signaling pathway 6.01x10-3 Environmental information Processing 4 NF-kappa B signaling pathway 2.27x10-9 6 Cell adhesion molecules (CAMs) 2.44x10-6 12 Cytokine- cytokine receptor interaction 9.62x10-4 Green boxes represent query genes linked to the pathway; Purple boxes indicate genes which are part of the pathway. Darker shades indicate higher connectivity. FWER: Family-Wise Error Rate. #: Order number. The results are sorted by increasing FWER. 5 1 1 4 1 4 4 3 2 3 1 5 1 1 1 7 3 5 2 1 1 7 2 1 4 2 1 Page 38 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Figure 1 189x113mm (300 x 300 DPI) Page 77 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Figure 2 189x214mm (300 x 300 DPI) Page 78 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Figure 3 189x257mm (300 x 300 DPI) Page 79 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Figure 4 189x279mm (300 x 300 DPI) Page 80 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Figure 5 189x126mm (300 x 300 DPI) Page 81 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Figure 6 189x96mm (300 x 300 DPI) Page 82 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Figure 7 189x158mm (300 x 300 DPI) Page 83 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Figure 8 189x243mm (300 x 300 DPI) Page 84 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 1 SUPPLEMENTARY FIGURE CAPTIONS Supplementary Figure S1.- Human C4BP(-) down-regulates CD80 and CD86 co- stimulatory molecules in mouse BMDCs stimulated through the TLR7 agonist gardiquimod. Mouse BMDCs, generated as described in Supplementary Methods, were incubated throughout their differentiation and maturation process with 5 g/ml of human C4BP(-). BMDC maturation was achieved by gardiquimod treatment (10 g/ml). Cells were then collected, washed, and analyzed by flow cytometry for cell surface expression of CD80 and CD86 surface markers. (a) Histograms from one representative experiment are shown. The MFIs for the cell surface markers are indicated in each histogram. (b) MFI for CD80 and CD86 cell surface markers. iDC, untreated, immature BMDCs; mDC, untreated, gardiquimod-matured BMDCs; C4BP(-), C4BP(-)-treated, gardiquimod-matured BMDCs. The results shown are the median +/- IQR from 3 independent experiments (*p < 0.05 compared to mDC). Supplementary Figure S2.- Time-course of anti-human C4BP(-) antibody development in lupus-prone NZBWF1 mice. Serum levels of anti-human C4BP(-) antibody production were measured by ELISA from week 20 (pretreatment) to week 36. The anti-human C4BP(-) antibody titer increased progressively with the time in the C4BP(-)-treated mice. Cyclophosphamide (CYP), used as a negative control group, did not develop anti-human C4BP(-)-specific antibodies at any time of the study period. Data are expressed as mean O.D. (450 nm) + SD (n= 6 mice/group). ****p < 0.0001 compared with CYP-treated mice. Supplementary Figure S3.- Renal function determination in lupus-prone NZBW F1 mice after subcutaneous administration low-dose rC4BP(-). (a) Schematic administration Page 85 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 2 schedule and dosage for subcutaneous (sc) injection of C4BP(-) and vehicle (PBS) (blue arrows), and cyclophosphamide (CYP) (red arrows) in NZBW F1 mice. (b) Unless otherwise indicated, proteinuria was monitored monthly over the course of the assay until week 37. Total 24-h urinary protein was determined by Pyrogallol Red-molybdate protein dye-binding assay. Data are normalized by mouse weight and expressed as mean values + SD (n= 6 mice/group); *p < 0.05; ***p < 0.001; ****p < 0.0001 compared with control, PBS-treated mice. (c) Time-course of anti-human rC4BP(-) antibody development from rC4BP(-)-treated NZBWF1 mice. Serum levels of anti-human rC4BP(-) antibody production were measured by ELISA from week 21 (pretreatment) to week 37. The anti-human rC4BP(-) antibody titer increased progressively with the time in the rC4BP(-)-treated mice. Cyclophosphamide (CYP), used as a negative control group, did not develop anti-human rC4BP(-)-specific antibodies at any time of the study period. Data are expressed as mean O.D. (450 nm) + SD (n= 6 mice/group). ****p < 0.0001 compared with CYP-treated mice. (d) Kaplan-Meier survival curves from lupus-prone NZBWF1 mice. Cumulative survival curves showed 100% survival in the CYP-treated mice (6/6), and 67% survival (4/6) in C4BP(-)-treated mice at the end of the study (day 337), while all vehicle PBS- treated mice died by day 313 (0/6). Red arrows identify the start (day 168) and the end (day 252) of the treatment period (n=6 mice/group); *p < 0.05; ****p < 0.0001, compared with vehicle PBS-treated mice; long-rank test. Supplementary Figure S4.- Comparative LN pathology determination in lupus-prone NZBW F1 mice after administration of both C4BP isoforms: C4BP(+) and C4BP(-). (a) Schematic administration schedule and dosage for intraperitoneal (ip) injection of C4BP(+), C4BP(-) and vehicle (PBS) (blue arrows) in NZBW F1 mice. (b) Proteinuria was monitored monthly over the course of the assay from week 21 until week 35. Total 24-h urinary protein was determined by Pyrogallol Red-molybdate protein dye-binding assay. Data are normalized by Page 86 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 3 mouse weight and expressed as cumulative mean values (n= 8 mice/group); *p < 0.05 compared with C4BP(+)-treated and control, PBS-treated mice. (c) Blood urea nitrogen (BUN) levels were assessed by a coupled enzyme reaction both before (week 21) and during (weeks 29 and 33) C4BP treatment. Data are expressed as mean + SD (n= 4 mice/group); *p < 0.05; **p < 0.01, compared with C4BP(+)-treated mice (w33). (d) Serum levels of anti-dsDNA antibodies correlating with disease activity were measured by ELISA 1 week and 5 weeks after the start of the treatment (weeks 25 and 29). Data are expressed as mean + SD (n= 3 mice/group); *p < 0.05 compared with C4BP(+)-treated mice (w25); *p < 0.05 compared with vehicle PBS-treated mice (w29). (e) Kaplan-Meier survival curves from lupus-prone NZBW F1 mice. Cumulative survival curves showed 100% survival (8/8) in the C4BP(-)-treated mice, 75% survival (6/8) in PBS-treated mice, and 62.5% survival (5/8) in PBS-treated mice at the end of the study (day 252). Red arrows identify the start (day 168) and the end (day 252) of the treatment period (n= 8 mice/group; *p = 0.06), compared with C4BP(+)-treated mice; long-rank test. (f) Representative sections of renal cortex from 36-week old mice treated with C4BP(+) (left), C4BP(-) (center) and vehicle PBS (right) mice stained with hematoxylin and eosin. Images are shown at 200 m magnification. The data obtained were used to semi-quantitatively grade histopathology traits indicating active LN (mesangial expansion, endocapillary proliferation, glomerular deposits, extracapillary proliferation and interstitial infiltrates), as well as chronic lesions (tubular atrophy and interstitial fibrosis) (n= 3 mice/group). Supplementary Figure S5.- Effect of C4BP(+) and C4BP(-) treatment in the activity of the classical pathway of complement from NZBW F1 mouse sera. The activity of the classical pathway of complement was measured as deposition of C3b on antibody-opsonized K562 cells and incubated with mouse sera for 30 min at 37 ºC. Deposited C3b was detected using a FITC-labelled antibody and flow cytometry. MFI, median fluorescence intensity. Page 87 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 4 Complement activity levels of NZW F1 mice analyzed before the start of C4BP treatment (week 21; n= 6 mice/group) and throughout the treatment (weeks 25 (n= 6 mice/group), 29 and 33 (n= 3 mice/group)). Data are expressed as mean + SD. *p < 0.05 compared with both C4BP(+)- treated and PBS-treated mice. Serum from C57BL6 mice (14-week old; n= 4) was included as a reference control for normal complement activity. Negative controls (Inact.) from all analyzed samples were determined after serum inactivation at 56 ºC for 30 min. Supplementary Figure S6.- Electrophoretic analysis of plasma-purified human C4BP(+) and C4BP(-) isoforms. Both plasma-purified human C4BP(+) and C4BP(-) underwent 3- 8% gradient SDS-PAGE under non-reducing (NR) and reducing (R) conditions. Under NR conditions, C4BP(-) is an homooligomer composed of 7 identical -chains, and C4BP(+) is an heterooligomer composed of 7 identical -chains and a unique -chain. All chains are covalently linked by their C-termini forming a spider-like structure in both isoforms, which migrate as a 500-570 kDa band (upper arrow). The -chain is always in complex with the vitamin K- dependent anticoagulant PS (70 kDa). Thus, under reducing conditions, the diffuse 70 kDa band (lower arrow) corresponds to the disassembled -chains in the case of the C4BP(-) isoform, and to the disassembled PS plus the -chains in the case of the C4BP(+) isoform. MW marker: molecular weight marker. Page 88 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 5 SUPPLEMENTARY METHODS Proteins and drugs C4BP(-) purification from human plasma (40 liters) involved BaCl2 precipitation followed by four sequential chromatography steps including heparin chromatography, hydrophobic interaction (butyl) chromatography, anion exchange (Q Sepharose) chromatography and, finally, size exclusion (Superdex) chromatography. Analogously, Recombinant C4BP(-) (rC4BP(-)) was transiently produced in HEK293cells (Expi293 cells) and purified from the cell culture supernatants through an hydrophobic interaction (butyl) chromatography followed by an anion exchange (Q Sepharose) chromatography, according to Bioingenium protocols (Bioingenium, Barcelona, Spain). All C4BP isoforms (plasma-purified C4BP(+) and C4BP(-), and rC4BP(-)) were concentrated, dialyzed and recovered in PBS buffer, pH 7.4. The purity of C4BP glycoproteins was higher than 85%, as assessed by Tris-Acetate 3-8% SDS-PAGE (NuPAGE precast protein gels; ThermoFisher, Waltham, MA, USA) from 6 g protein/lane, and further Coomassie Blue staining (Supplementary Figure S6, and data not shown). All C4BP proteins employed in the studies were endotoxin-free, as assessed through the Limulus amebocyte lysate test (GenScript, Piscataway, NJ, USA). CYP (Genoxal®, Baxter Oncology GmbH, Halle/Westfallen, Germany), was resuspended in saline and administered at a dose of 2.5 mg in a final volume of 0.13 ml. Bone marrow-derived DC (BMDC) generation Femurs and tibiae of female, 14 weeks-old BALB/c mice (Charles River Laboratories, Wilmington, MA, USA) were removed and purified from the surrounding muscle tissue by Page 89 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 6 rubbing with sterile tissues and placed in RPMI-1640 medium (Gibco, ThermoFisher). Thereafter intact bones were soaked in 70% ethanol for desinfection. Both ends were cut with scissors and the marrow was flushed with RPMI-1640 medium, using a 1-ml insulin syringe with a 25G needle, onto a cell strainer (BD Biosciences, Bedford, MA, USA) to obtain a uniform single-cell suspension. 1x106 viable bone marrow cells were resuspended in RPMI 1640 supplemented with 100 mg/ml streptomycin, 100 IU/ml penicillin, 2 mM L-glutamine (all from ThermoFischer) and 10% heat-inactivated FBS (Cultek, Madrid Spain) and plated in 60-mm culture plates at 37ºC under 5% CO2. For surface phenotype determination, BMDCs were generated supplementing cultures with rmGM-CSF (20ng/ml) (Peprotech, London, UK) at days 0, 3 and 6 of culture. C4BP (β-) was added at 5 μg/ml at days 0 and 3 of culture. At day 6, non- adherent cells were re-seeded into 24-well plates with fresh medium at 5x105 cells/ml and were further stimulated for 24h with 10 g/ml Gardiquimod (InvivoGen, San Diego, CA, USA). Mice, study design and follow up Both NZBW F1 and MRL-lpr mouse strains (Jackson Laboratory, Bar Harbor, ME, USA) develop spontaneously an autoimmune disease resembling human SLE. The animals were maintained under standard laboratory conditions, at 20-24 ºC and 40-70% relative humidity, with 12-hour fluorescent light/12-hour dark cycle. They were feed standard diet and tap water ad libitium. The selection of the highest C4BP(-) dose for in vivo administration was based in previous studies employing complement-related proteins in murine models of immune- inflammatory pathologies.S1–S3 We opted for the beginning of C4BP(-) and C4BP(+) treatments at 24 weeks because it has been reported that when 23 weeks old, while histologically normal, NZBW F1 mice already show faint staining of the glomeruli with anti-IgG and increased expression of several inflammatory markers.S4 Analogously, at the beginning of C4BP(-) Page 90 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 7 administration, 10 week old MRL-lpr mice were fully hypocomplementemic (data not shown), indicative of high levels of circulating ICs and active disease.S5 Neither toxicity nor behavioral changes were observed in the mice as a consequence of C4BP(-), C4BP(+) or CYP administration. Body weight was determined twice monthly (NZBW F1 mice) or weekly (MRL-lpr mice) from the beginning to the end of follow-up. Mice were placed in metabolic cages to collect 24 h urine specimens before the onset of treatment and monthly (NZBW F1 mice) or biweekly (MRL-lpr mice) thereafter. Blood was obtained from the tail vein at monthly intervals and at the sacrifice. Kidneys were dissected and processed for histological, biochemical and molecular analyses at the end of the study. In MRL-lpr mice, macroscopic SLE-like skin lesions from the interscapular region, the snout and the ears were scored weekly in a semiquantitative manner using a 0 to 4 scoring system: 0, no visible skin changes; 1, minimal hair loss with redness and a few scattered lesions; 2, redness and hair loss with a small area of involvement (< 0,5 cm2); 3, redness, scabbing, and lesion(s) with total area > 0.5, but < 1.0 cm2; and 4, redness, scabbing, and lesion(s) with total area > 1.0 cm2. All experiments were carried out in accordance with current EU legislation on animal experimentation and were approved by ‘‘CEEA: Animal Experimentation Ethics Committee”, the Institutional Ethics University of Barcelona Committee for Animal Research, and the Generalitat de Catalunya (DARP: 8765). ELISA for anti-human C4BP(-) detection To assess the development of antibodies against administered human rC4BP(-) by the NZBW F1 mice, we set up an indirect ELISA assay. Briefly, we immobilized C4BP(-) (200 ng/100 l/well, dissolved in coating buffer: 100 mM Na2CO3 / NaHCO3, pH 9.6) by direct adsorption in a 96-well plate overnight at 4 ºC. Next, the plate was blocked with Tris-Tween (50 Page 91 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 8 mM Tris pH 7.4 + 150 mM NaCl + 0.2% Tween 20) + 1% BSA (100 ml/well) for 1h at room temp. The collected mouse sera were diluted to 1:500 in Tris-Tween + 1% BSA and incubated in duplicate to the plate (100 l/well) for 1.5 h at room temp. Human C4BP(-)-specific antibodies were detected by incubation with an anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody (Agilent Technologies, Santa Clara, CA, USA) (1:10,000 dilution) in Tris- Tween + 1% BSA for 30 min. at room temp. Washing between steps was performed with Tris- Tween. Finally, the plate was developed with TMB (3,3’,5,5’-tetramethylbenzidine) HRP substrate, stopped with sulfuric acid (1N H2SO4) and the absorbances were read at 450 nm in a microtiter plate reader. Negative controls included sera from CYP- and PBS-treated mice. Flow cytometry Cell surface phenotypes were analyzed using FITC-conjugated anti–CD80 (16-10A1) and PE-conjugated anti-CD86 (PO3.3) mAbs (all from Miltenyi Biotec, Bergisch Gladbach, Germany). After washing with PBS, cells were subsequently stained with 3μl mAbs/105 cells in 100 l FACS buffer (PBS containing 1% BSA and 0.1% sodium azide) for 15 min at room temperature. To exclude debris, BMDCs were gated according to forward scatter (FSC) and side scatter (SSC) parameters. Staining with 7-aminoactinomycin D (ThermoFischer) was also employed to assess the viability status of BMDCs. Stained cells were analyzed using a FACSCanto II flow cytometer equipped with FACSDiva software (Becton Dickinson, Franklin Lakes, NJ, USA). Subsequent analyses were performed through FlowJo software (Flowjo LLC, Ashland, OR, USA). Renal histopathology Page 92 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 9 To determine the extent of renal damage, all sections were analyzed by two blinded pathologists. Typical glomerular active lesions of LN were evaluated: mesangial expansion, endocapillary proliferation, glomerular deposits, extracapillary proliferation and interstitial infiltrates, as well as tubulointerstitial chronic lesions: tubular atrophy and interstitial fibrosis. Lesions were graded semi-quantitatively using a four-point scoring system: (-) null, (+) low, (++) moderate, (+++) severe. Glomerular cross-sectional area (µm2) was calculated based on average area of 150 glomeruli in each group, from digitized images taken at x100 (3 mice/group), measured using ImageJ v1.52c software (http://fiji.sc/; NIH, Bethesda, MD, USA). Immunofluorescent determination of renal IC and complement deposition Kidney slices were fixed in 4% paraformaldehyde, embedded in Tissue Tec OCT compound (Sakura, Alpen aan den Rijn, Netherlands) and stored at -80 ºC. Five-m cryostat sections were used for confocal microscopy to quantify the mean fluorescence intensity of the Cy5.5 fluorochrome. At least 10 glomeruli per section were visualized and photographed with an immunofluorescence confocal microscope Leica TCS-SL spectral (Leica Microsystems GmbH, Wetzlar, Germany). Fluorescence was quantified with Leica software and expressed as mean fluorescence intensity (MFI). Complement activity assay The activity of the classical pathway of complement was determined by C3b deposition on K562 cells (ATCC, LGC Standards, Barcelona, Spain) opsonized with rabbit polyclonal antibodies (Agrisera, Vännäs, Sweden). Briefly, K562 cells were cultured in suspension in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated FBS, 100 Page 93 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 10 mg/ml streptomycin, 100 IU/ml penicillin, 2 mM L-glutamine (all from ThermoFischer). The cells were washed twice in cold PBS, and 106 cells were added to reaction tubes containing PBS with 2 mM MgCl2, 0.15 mM CaCl2, 5 mg/ml of opsonizing antibodies and 10% mouse sera in a total volume of 100 μl. After 30 min incubation at 37ºC, cells were washed with cold FACS buffer. The amount of deposited C3b was measured using FITC-conjugated goat IgG fraction to mouse complement C3 antibody (MP Biomedicals, Solon, OH, USA) diluted 1:100, and allowed to bind for 1 h at 4ºC. The cells were washed three times, resuspended with cold FACS buffer, and analyzed in a FACSCanto II flow cytometer equipped with FACSDiva software (Becton Dickinson). Immunohistochemistry Paraffin-processed sections (5 µm) were deparaffinized in xylene, rehydrated in graded ethanol solutions, rinsed in distilled water and treated with 3% hydrogen peroxide in methanol (30 min at room temp. (RT)) to remove endogenous peroxidase activity. The sections were further incubated 30 min at RT with blocking solution (5% goat serum, 0.1% Tween 20 in PBS, pH 7.2), and incubated overnight at 4ºC with the primary antibodies, washed in PBS and incubated with the appropriate secondary antibodies for 30-60 min at RT. Colour was developed using 3,3’-diaminobenzidine (DAB) and sections were counterstained with hematoxylin before dehydration, clearing, and mounting. Negative controls in which the primary antibody was replaced with PBS were used to test for non-specific binding (data not shown). TUNEL Assay Briefly, 5 μm thick deparaffinized sections were pretreated with proteinase K for 30 min at RT. After washing in deionized water, the endogenous peroxidase was inactivated using 3% Page 94 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 11 H2O2 in methanol for 5 min, followed by incubation with TdT enzyme at 37°C for 1 h. Next, sections were immersed in TdT Stop buffer and incubated with Streptavidin-HRP solution at 37 ºC for 10 min followed by DAB solution. Slices were counterstained with 1% methyl green. TUNEL-positive cells were assessed by light microscopy. The number of cortical apoptotic cells was quantified in 10 fields from 3 section samples/group and given as apoptotic cells/field. Ten glomeruli per section (30 glomeruli/group) were also quantified to establish the number of apoptotic cells per glomerular cut section (cells/gcs). ImageJ v1.52c software (NIH) was employed to analyze digitized images at x200 and x400, respectively. Differential gene expression analysis Total RNA from mouse kidneys was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the High-capacity cDNA archive kit (Applied BioSystems, Carlsbad, CA, USA). Gene expression profiling was performed in 384- well microfluidic cards pre-loaded with 377 gene expression assays for transcripts relevant in murine LN. S6-S8 Four wells per card included the 18S gene expression assay as internal control. We used one card for each pool (2-3 mice/pool; 3 cards/group). Data were obtained with SDS v2.4 and RQ Manager v1.2.1, analyzed with DataAssist software v3.0 (all from Applied Biosystems) and normalized using 3 endogenous controls (Actb, Gapdh and Gusb). The values obtained were relativized using the vehicle PBS-treated group as reference. Differentially expressed genes were analyzed using the Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, Qiagen) to identify biological and molecular networks differentially regulated in the LN model. IPA core analysis is a source of gene-interaction based pathway analysis including canonical pathways and a knowledge database based on scientific findings. Differentially expressed genes at least 1.8-fold upregulated or downregulated were imported and analyzed in the IPA database. Based on the direct or indirect connectivity of genes as disclosed Page 95 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 12 in the literature, genes were mapped onto biological pathways and disease networks. A P value of < 0.05 (Fisher exact test) was used as the cutoff for significant biological functions, networks, and pathways, and they were ranked by ratio. Downstream effects analysis was undertaken to identify key biological processes influenced by differentially expressed genes. Statistical significance of the overlap between two groups of genes from our dataset and from the “Ingenuity® Knowledge Base” was given as overlap p-value, and significant process or pathway directionality was predicted when 2 < Z-score < -2. Selected gene transcripts were further validated in individual mouse kidney samples by RT-qPCR using the corresponding inventoried TaqMan Gene Expression Assays (Applied BioSystems). Quantification was achieved through the ΔΔCt method. A relative fold change in mRNA abundance was calculated with the equation 2-ΔΔCt, employing Gusb as endogenous reference transcript. Pathway annotation of the RT-qPCR-validated gene set was performed through PathwAX, which uses the comprehensive network FunCoup to analyze network crosstalk between a query gene list and KEGG pathways. Mouse cytokine array Serum was obtained by centrifugation of blood samples at sacrifice (1500 g, 10 min, 4ºC). Tissue lysates were obtained by kidney homogenization in PBS supplemented with the protease inhibitors aprotinin (Sigma-Aldrich), leupeptin and pepstatin (both from Tocris Bioscience, Bristol, UK) (all 10μg/ml) using a Dounce Homogenizer (Knotes Glass, Vineland, NJ, USA). Triton X-100 was added to a final concentration of 1% before the lysates underwent freezing at -80°C. After thawing, the lysates were centrifuged at 10000 g for 5 min. Pooled samples (100 l serum, or 300 g protein extracts; 2 mice/group) were analyzed using the Proteome Profiler Array “Mouse Cytokine Array Panel A” kit (R&D Systems), according to the Page 96 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 13 manufacturer’s instructions, to determine the relative levels of 40 cytokines and chemokines and acute phase proteins. Page 97 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 14 References S1. Blom AM, Nandakumar KS, Holmdahl R. C4b-binding protein (C4BP) inhibits development of experimental arthritis in mice. Ann Rheum Dis. 2009;68:136-142. S2. Rodriguez W, Mold C, Marnell LL, et al. Prevention and reversal of nephritis in MRL/lpr mice with a single injection of C-reactive protein. Arthritis Rheum. 2006;54:325-335. S3. Fakhouri F, de Jorge EG, Brune F, et al. Treatment with human complement factor H rapidly reverses renal complement deposition in factor H-deficient mice. Kidney Int. 2010;78:279-286. S4. Schiffer L, Bethunaickan R, Ramanujam M, et al. Activated renal macrophages are markers of disease onset and disease remission in lupus nephritis. J Immunol. 2008;180:1938-1947. S5 Andrews BS, Eisenberg RA, Theofilopoulos AN, et al. Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains. J Exp Med. 1978; 148:1198-1215. S6. Berthier CC, Bethunaickan R, Gonzalez-Rivera T, et al. Cross-species transcriptional network analysis defines shared inflammatory responses in murine and human lupus nephritis. J Immunol. 2012;189:988-1001. S7. Bethunaickan R, Berthier CC, Zhang W, et al. Comparative transcriptional profiling of 3 murine models of SLE nephritis reveals both unique and shared regulatory networks. PLoS ONE 2013;8:e77489. S8. Bethunaickan R, Berthier CC, Zhang W, et al. Identification of stage-specific genes associated with lupus nephritis and response to remission induction in (NZB x NZW)F1 and NZM2410 mice. Arthritis Rheum. 2014;66:2246-2258 Page 98 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 15 Table S1.- List of relevant SLE genes included in the customized TaqMan Low Density Array. Gene Symbol Assay ID UniGene ID Gene name Acad8 Mm00482266_m1 Mm.289244 acyl-Coenzyme A dehydrogenase family, member 8 Ace Mm00802048_m1 Mm.754 angiotensin I converting enzyme (peptidyl-dipeptidase A) 1 Acsl1 Mm00484217_m1 Mm.210323 acyl-CoA synthetase long-chain family member 1 Acsl3 Mm01255804_m1 Mm.276016 acyl-CoA synthetase long-chain family member 3 Actr3 Mm02342769_g1 Mm.183102 ARP3 actin-related protein 3 Ada Mm00545720_m1 Mm.388 adenosine deaminase Adh1 Mm00507711_m1 Mm.2409 alcohol dehydrogenase 1 (class I) Adh7 Mm00507750_m1 Mm.8473 alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide Adhfe1 Mm00613830_m1 Mm.28514 alcohol dehydrogenase, iron containing, 1 Adsl Mm00507759_m1 Mm.38151 adenylosuccinate lyase Agt Mm00599662_m1 Mm.301626 angiotensinogen (serpin peptidase inhibitor, clade A, member 8) Ahcyl1 Mm00461101_m1 Mm.220328 S-adenosylhomocysteine hydrolase-like 1 Ahcyl2 Mm00619649_m1 Mm.210899 S-adenosylhomocysteine hydrolase-like 2 Aifm1 Mm00442540_m1 Mm.240434 apoptosis-inducing factor, mitochondrion-associated 1 Aim2 Mm01295719_m1 Mm.131453 absent in melanoma 2 Akr1a1 Mm00480608_m1 Mm.30085 aldo-keto reductase family 1, member A1 (aldehyde reductase) Aldh2 Mm00477463_m1 Mm.284446 aldehyde dehydrogenase 2, mitochondrial Aldh3a2 Mm00839320_m1 Mm.398221 aldehyde dehydrogenase family 3, subfamily A2 Aldh3b1 Mm00550698_m1 Mm.109341 aldehyde dehydrogenase 3 family, member B1 Aldh4a1 Mm00615268_m1 Mm.273571 aldehyde dehydrogenase 4 family, member A1 Aldh6a1 Mm00506227_m1 Mm.247510 aldehyde dehydrogenase family 6, subfamily A1 Aldh7a1 Mm00519645_m1 Mm.30250 aldehyde dehydrogenase family 7, member A1 Aldh9a1 Mm00480240_m1 Mm.292539 aldehyde dehydrogenase 9, subfamily A1 Apoc3 Mm00445670_m1 Mm.390161 apolipoprotein C-III Apoe Mm01307193_g1 Mm.305152 apolipoprotein E Arf6 Mm00500208_s1 Mm.27308 ADP-ribosylation factor 6 Arg1 Mm00475988_m1 Mm.154144 arginase, liver Arg2 Mm00477592_m1 Mm.3506 arginase type II Arpc1b Mm00834862_m1 Mm.30010 actin related protein 2/3 complex, subunit 1B Arpc2 Mm01254383_m1 Mm.337038 actin related protein 2/3 complex, subunit 2 Arpc3 Mm01199871_m1 Mm.275942 actin related protein 2/3 complex, subunit 3 Arpc5 Mm04208715_m1 Mm.288974 actin related protein 2/3 complex, subunit 5 Atf4 Mm00515325_g1 Mm.641 activating transcription factor 4 Atp5b Mm00443967_g1 Mm.238973 ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit Atp5f1 Mm01296543_g1 Mm.251152 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit B1 Atp5g3 Mm01334541_g1 Mm.2966 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 (subunit 9) Auh Mm00479363_m1 Mm.252034 AU RNA binding protein/enoyl-coenzyme A hydratase B2m Mm00437762_m1 Mm.163 beta-2 microglobulin Batf3 Mm01318274_m1 Mm.6922 basic leucine zipper transcription factor, ATF-like 3 Bckdha Mm00476112_m1 Mm.25848 branched chain ketoacid dehydrogenase E1, alpha polypeptide Bckdhb Mm01177077_m1 Mm.12819 branched chain ketoacid dehydrogenase E1, beta polypeptide Page 99 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 16 Blk Mm00432077_m1 Mm.3962 B lymphoid kinase Btk Mm00442712_m1 Mm.4475 Bruton agammaglobulinemia tyrosine kinase C1qa Mm00432142_m1 Mm.439957 complement component 1, q subcomponent, alpha polypeptide C1qb Mm01179619_m1 Mm.2570 complement component 1, q subcomponent, beta polypeptide C1qc Mm00776126_m1 Mm.439732 complement component 1, q subcomponent, C chain C1ra Mm04206253_g1 Mm.333375 complement component 1, r subcomponent A C1s1 Mm00663210_mH Mm.219527 complement component 1, s subcomponent 1 C3 Mm00437838_m1 Mm.19131 complement component 3 C3ar1 Mm02620006_s1 Mm.2408 complement component 3a receptor 1 C4bp Mm00432150_m1 Mm.306720 complement component 4 binding protein C6 Mm00489521_m1 Mm.20247 complement component 6 C8a Mm00521627_m1 Mm.197638 complement component 8, alpha polypeptide C9 Mm00442739_m1 Mm.29095 complement component 9 Casp1 Mm00438023_m1 Mm.1051 caspase 1 Casp3 Mm01195085_m1 Mm.34405 caspase 3 Cat Mm00437992_m1 Mm.4215 catalase Cbs Mm00460654_m1 Mm.206417 cystathionine beta-synthase Ccl2 Mm00441242_m1 Mm.290320 chemokine (C-C motif) ligand 2 Ccl5 Mm01302427_m1 Mm.284248 chemokine (C-C motif) ligand 5 Ccl9 Mm00441260_m1 Mm.416125 chemokine (C-C motif) ligand 9 Ccr1 Mm00438260_s1 Mm.274927 chemokine (C-C motif) receptor 1 Ccr2 Mm00438270_m1 Mm.6272 chemokine (C-C motif) receptor 2 Ccr5 Mm01963251_s1 Mm.14302 chemokine (C-C motif) receptor 5 Ccr6 Mm99999114_s1 Mm.8007 chemokine (C-C motif) receptor 6 Ccr7 Mm01301785_m1 Mm.2932 chemokine (C-C motif) receptor 7 Ccs Mm00444148_m1 Mm.434411 copper chaperone for superoxide dismutase Cd14 Mm00438094_g1 Mm.3460 CD14 antigen Cd19 Mm00515420_m1 Mm.4360 CD19 antigen Cd1d1 Mm00783541_s1 Mm.1894 CD1d1 antigen Cd207 Mm00523545_m1 Mm.136079 CD207 antigen Cd22 Mm00515432_m1 Mm.260994 CD22 antigen Cd274 Mm00452054_m1 Mm.245363 CD274 antigen Cd276 Mm00506020_m1 Mm.5356 CD276 antigen Cd28 Mm00483137_m1 Mm.255003 CD28 antigen Cd300lb Mm01701741_m1 Mm.185355 CD300 antigen like family member B Cd3d Mm00442746_m1 Mm.4527 CD3 antigen, delta polypeptide Cd3g Mm00438095_m1 Mm.335106 CD3 antigen, gamma polypeptide Cd40 Mm00441891_m1 Mm.271833 CD40 antigen Cd40lg Mm00441911_m1 Mm.4861 CD40 ligand Cd48 Mm00455932_m1 Mm.1738 CD48 antigen Cd5 Mm00432417_m1 Mm.779 CD5 antigen Cd74 Mm00658576_m1 Mm.439737 CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated) Cd79b Mm00434143_m1 Mm.2987 CD79B antigen Cd80 Mm00711660_m1 Mm.89474 CD80 antigen Cd83 Mm00486868_m1 Mm.57175 CD83 antigen Cd86 Mm00444543_m1 Mm.1452 CD86 antigen Page 100 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 17 Cdt1 Mm00466006_m1 Mm.21873 chromatin licensing and DNA replication factor 1 Cebpb Mm00843434_s1 Mm.439656 CCAAT/enhancer binding protein (C/EBP), beta Cfb Mm00433918_g1 Mm.653 complement factor B Cfh Mm01299248_m1 Mm.8655 complement component factor h Cfi Mm00432470_m1 Mm.117180 complement component factor i Chek2 Mm00443839_m1 Mm.279308 checkpoint kinase 2 Chga Mm00514341_m1 Mm.4137 chromogranin A Cldn1 Mm00516701_m1 Mm.289441 claudin 1 Cldn7 Mm00516817_m1 Mm.281896 claudin 7 Clec10a Mm00546125_g1 Mm.252405 C-type lectin domain family 10, member A Clec4n Mm00490934_m1 Mm.271782 C-type lectin domain family 4, member n Clec7a Mm01183349_m1 Mm.239516 C-type lectin domain family 7, member a Clu Mm01197002_m1 Mm.200608 clusterin Col18a1 Mm00487131_m1 Mm.4352 collagen, type XVIII, alpha 1 Col1a1 Mm00801666_g1 Mm.277735 collagen, type I, alpha 1 Col1a2 Mm00483888_m1 Mm.277792 collagen, type I, alpha 2 Col3a1 Mm01254476_m1 Mm.249555 collagen, type III, alpha 1 Col4a1 Mm01210125_m1 Mm.738 collagen, type IV, alpha 1 Cox11 Mm01615963_g1 Mm.151940 cytochrome c oxidase assembly protein 11 Cox15 Mm00523096_m1 Mm.248237 cytochrome c oxidase assembly protein 15 Cox5a Mm01176957_g1 Mm.273403 cytochrome c oxidase subunit Va Cox5b Mm00833840_g1 Mm.180182 cytochrome c oxidase subunit Vb Cox6a1 Mm01612194_m1 Mm.43415 cytochrome c oxidase subunit VIa polypeptide 1 Cox6b1 Mm00824357_m1 Mm.400 cytochrome c oxidase, subunit VIb polypeptide 1 Cox8a Mm02342396_g1 Mm.14022 cytochrome c oxidase subunit VIIIa Cpeb1 Mm01314928_m1 Mm.273122 cytoplasmic polyadenylation element binding protein 1 Csf1 Mm00432686_m1 Mm.795 colony stimulating factor 1 (macrophage) Csf1r Mm01266652_m1 Mm.22574 colony stimulating factor 1 receptor Csf2rb Mm00655745_m1 Mm.235324 colony stimulating factor 2 receptor, beta, low-affinity (granulocyte- macrophage) Cst7 Mm00438349_m1 Mm.12965 cystatin F (leukocystatin) Ctgf Mm01192932_g1 Mm.390287 connective tissue growth factor Ctla4 Mm00486849_m1 Mm.390 cytotoxic T-lymphocyte-associated protein 4 Ctss Mm01255859_m1 Mm.3619 cathepsin S Cx3cl1 Mm00436454_m1 Mm.103711 chemokine (C-X3-C motif) ligand 1 Cx3cr1 Mm02620111_s1 Mm.44065 chemokine (C-X3-C motif) receptor 1 Cxcl1 Mm04207460_m1 Mm.21013 chemokine (C-X-C motif) ligand 1 Cxcl10 Mm00445235_m1 Mm.877 chemokine (C-X-C motif) ligand 10 Cxcl11 Mm00444662_m1 Mm.131723 chemokine (C-X-C motif) ligand 11 Cxcl13 Mm04214185_s1 Mm.10116 chemokine (C-X-C motif) ligand 13 Cxcl16 Mm00469712_m1 Mm.425692 chemokine (C-X-C motif) ligand 16 Cxcl2 Mm00436450_m1 Mm.4979 chemokine (C-X-C motif) ligand 2 Cxcl9 Mm00434946_m1 Mm.766 chemokine (C-X-C motif) ligand 9 Cxcr4 Mm01996749_s1 Mm.1401 chemokine (C-X-C motif) receptor 4 Cxcr6 Mm02620517_s1 Mm.124289 chemokine (C-X-C motif) receptor 6 Cyc1 Mm00470540_m1 Mm.29196 cytochrome c-1 Dbf4 Mm01324087_m1 Mm.292470 DBF4 homolog (S. cerevisiae) Page 101 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 18 Ddr1 Mm01273496_m1 Mm.5021 discoidin domain receptor family, member 1 Dhrs4 Mm00472717_m1 Mm.27427 dehydrogenase/reductase (SDR family) member 4 Dld Mm00432831_m1 Mm.3131 dihydrolipoamide dehydrogenase Dnaja1 Mm00787254_s1 Mm.27897 DnaJ (Hsp40) homolog, subfamily A, member 1 Egln3 Mm00472200_m1 Mm.133037 egl-9 family hypoxia-inducible factor 3 Eng Mm00468256_m1 Mm.225297 endoglin Ep300 Mm00625535_m1 Mm.258397 E1A binding protein p300 Epas1 Mm01236112_m1 Mm.1415 endothelial PAS domain protein 1 Ephx2 Mm01313813_m1 Mm.15295 epoxide hydrolase 2, cytoplasmic Ereg Mm00514794_m1 Mm.4791 epiregulin F3 Mm00438853_m1 Mm.273188 coagulation factor III Fasl Mm00438864_m1 Mm.3355 Fas ligand (TNF superfamily, member 6) Fcer1g Mm02343757_m1 Mm.22673 Fc receptor, IgE, high affinity I, gamma polypeptide Fcgr1 Mm00438874_m1 Mm.150 Fc receptor, IgG, high affinity I Fcgr3 Mm00438882_m1 Mm.22119 Fc receptor, IgG, low affinity III Fcgr4 Mm00519988_m1 Mm.251254 Fc receptor, IgG, low affinity IV Fga Mm00802584_m1 Mm.88793 fibrinogen alpha chain Fgb Mm00805336_m1 Mm.30063 fibrinogen beta chain Fgg Mm00513575_m1 Mm.16422 fibrinogen gamma chain Fli1 Mm00484410_m1 Mm.258908 Friend leukemia integration 1 Fmo1 Mm00515795_m1 Mm.976 flavin containing monooxygenase 1 Fmo2 Mm00490159_m1 Mm.10929 flavin containing monooxygenase 2 Fmo4 Mm00467393_m1 Mm.155164 flavin containing monooxygenase 4 Fn1 Mm01256744_m1 Mm.193099 fibronectin 1 Fos Mm00487425_m1 Mm.246513 FBJ osteosarcoma oncogene Foxp3 Mm00475162_m1 Mm.182291 forkhead box P3 Fyn Mm00433373_m1 Mm.4848 Fyn proto-oncogene Got1 Mm01195792_g1 Mm.19039 glutamic-oxaloacetic transaminase 1, soluble Gpnmb Mm01328587_m1 Mm.302602 glycoprotein (transmembrane) nmb Gss Mm00515065_m1 Mm.252316 glutathione synthetase Gsto1 Mm00599866_m1 Mm.378931 glutathione S-transferase omega 1 Havcr1 Mm00506686_m1 Mm.17771 hepatitis A virus cellular receptor 1 Hck Mm01241463_m1 Mm.715 hemopoietic cell kinase Hmox1 Mm00516005_m1 Mm.276389 heme oxygenase 1 Hnf4a Mm01247712_m1 Mm.202383 hepatic nuclear factor 4, alpha Hspe1 Mm00434083_m1 Mm.215667 heat shock protein 1 (chaperonin 10) Icam1 Mm00516023_m1 Mm.435508 intercellular adhesion molecule 1 Icos Mm00497600_m1 Mm.42044 inducible T cell co-stimulator Id1 Mm00775963_g1 Mm.444 inhibitor of DNA binding 1 Idh1 Mm00516030_m1 Mm.9925 isocitrate dehydrogenase 1 (NADP+), soluble Idh3a Mm00499674_m1 Mm.279195 isocitrate dehydrogenase 3 (NAD+) alpha Idh3b Mm00504589_m1 Mm.29590 isocitrate dehydrogenase 3 (NAD+) beta Idh3g Mm00599686_g1 Mm.14825 isocitrate dehydrogenase 3 (NAD+), gamma Ifi202b Mm00839397_m1 Mm.218770 interferon activated gene 202B Ifi44 Mm00505670_m1 Mm.30756 interferon-induced protein 44 Ifih1 Mm00459183_m1 Mm.136224 interferon induced with helicase C domain 1 Page 102 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 19 Ifng Mm01168134_m1 Mm.240327 interferon gamma Igfbp1 Mm00515154_m1 Mm.21300 insulin-like growth factor binding protein 1 Igfbp6 Mm00599696_m1 Mm.358609 insulin-like growth factor binding protein 6 Ikbke Mm00444862_m1 Mm.386783 inhibitor of kappaB kinase epsilon Ikzf1 Mm01187882_m1 Mm.103545 IKAROS family zinc finger 1 Il10 Mm00439614_m1 Mm.874 interleukin 10 Il10ra Mm00434151_m1 Mm.379327 interleukin 10 receptor, alpha Il12a Mm00434165_m1 Mm.103783 interleukin 12a Il12b Mm00434174_m1 Mm.239707 interleukin 12b Il17a Mm00439618_m1 Mm.5419 interleukin 17A Il18 Mm00434225_m1 Mm.1410 interleukin 18 Il1b Mm00434228_m1 Mm.222830 interleukin 1 beta Il1f6 Mm00457645_m1 Mm.133095 interleukin 1 family, member 6 Il1r2 Mm00439629_m1 Mm.1349 interleukin 1 receptor, type II Il23a Mm01160011_g1 Mm.125482 interleukin 23, alpha subunit p19 Il27 Mm00461162_m1 Mm.222632 interleukin 27 Il27ra Mm00497259_m1 Mm.38386 interleukin 27 receptor, alpha Il2rg Mm00442885_m1 Mm.2923 interleukin 2 receptor, gamma chain Il33 Mm00505403_m1 Mm.182359 interleukin 33 Il4 Mm00445259_m1 Mm.276360 interleukin 4 Il7r Mm00434295_m1 Mm.389 interleukin 7 receptor Inpp5d Mm00494987_m1 Mm.15105 inositol polyphosphate-5-phosphatase D Irak1 Mm01193538_m1 Mm.38241 interleukin-1 receptor-associated kinase 1 Irf1 Mm01288580_m1 Mm.105218 interferon regulatory factor 1 Irf4 Mm00516431_m1 Mm.4677 interferon regulatory factor 4 Irf5 Mm00496477_m1 Mm.6479 interferon regulatory factor 5 Irf7 Mm00516793_g1 Mm.3233 interferon regulatory factor 7 Irf8 Mm00492567_m1 Mm.334861 interferon regulatory factor 8 Irf9 Mm00492679_m1 Mm.2032 interferon regulatory factor 9 Itga4 Mm01277951_m1 Mm.31903 integrin alpha 4 Itgam Mm00434455_m1 Mm.262106 integrin alpha M Itgb2 Mm00434513_m1 Mm.1137 integrin beta 2 Jak3 Mm00439973_m1 Mm.249645 Janus kinase 3 Jun Mm00495062_s1 Mm.275071 jun proto-oncogene Klk11 Mm00480210_g1 Mm.154276 kallikrein related-peptidase 11 Klkb1 Mm00434658_m1 Mm.482691 kallikrein B, plasma 1 Lat Mm00456761_m1 Mm.10280 linker for activation of T cells Lck Mm00802897_m1 Mm.293753 lymphocyte protein tyrosine kinase Lcp2 Mm01187570_m1 Mm.265350 lymphocyte cytosolic protein 2 Lrrk2 Mm00481934_m1 Mm.37558 leucine-rich repeat kinase 2 Ltb Mm00434774_g1 Mm.1715 lymphotoxin B Ly96 Mm01227593_m1 Mm.116844 lymphocyte antigen 96 Lyn Mm01217488_m1 Mm.317331 Yamaguchi sarcoma viral (v-yes-1) oncogene homolog Maob Mm00555412_m1 Mm.241656 monoamine oxidase B Map3k1 Mm00803707_m1 Mm.15918 mitogen-activated protein kinase kinase kinase 1 Map3k3 Mm00803725_m1 Mm.27041 mitogen-activated protein kinase kinase kinase 3 Page 103 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 20 Map3k8 Mm00432637_m1 Mm.3275 mitogen-activated protein kinase kinase kinase 8 Mapk9 Mm00444239_m1 Mm.68933 mitogen-activated protein kinase 9 Mat2b Mm00506137_m1 Mm.293771 methionine adenosyltransferase II, beta Mmp2 Mm00439498_m1 Mm.29564 matrix metallopeptidase 2 Mmp9 Mm00442991_m1 Mm.4406 matrix metallopeptidase 9 Mpo Mm01298424_m1 Mm.4668 myeloperoxidase Ms4a1 Mm00545909_m1 Mm.4046 membrane-spanning 4-domains, subfamily A, member 1 Msr1 Mm00446214_m1 Mm.239291 macrophage scavenger receptor 1 Myl9 Mm01251442_m1 Mm.271770 myosin, light polypeptide 9, regulatory Ncf1 Mm00447921_m1 Mm.425296 neutrophil cytosolic factor 1 Ndrg2 Mm00443481_g1 Mm.26722 N-myc downstream regulated gene 2 Ndufa10 Mm00600325_m1 Mm.248778 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10 Ndufa3 Mm01329704_g1 Mm.17851 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3 Ndufa8 Mm00503351_m1 Mm.19834 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8 Ndufa9 Mm00481216_m1 Mm.29939 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9 Ndufaf1 Mm00452828_m1 Mm.5390 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, assembly factor 1 Ndufb10 Mm01300078_m1 Mm.1129 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10 Ndufb2 Mm01157852_m1 Mm.29415 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 2 Ndufb5 Mm00452592_m1 Mm.28058 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5 Ndufb6 Mm01208591_g1 Mm.1103 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6 Ndufs1 Mm00523640_m1 Mm.290791 NADH dehydrogenase (ubiquinone) Fe-S protein 1 Ndufs5 Mm02600127_g1 Mm.42805 NADH dehydrogenase (ubiquinone) Fe-S protein 5 Ndufs7 Mm01144210_m1 Mm.28712 NADH dehydrogenase (ubiquinone) Fe-S protein 7 Nfatc3 Mm01249200_m1 Mm.383185 nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 3 Nfkb2 Mm00479810_g1 Mm.102365 nuclear factor of kappa light polypeptide gene enhancer in B cells 2, p49/p100 Nfkbie Mm01269649_m1 Mm.57043 nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, epsilon Nisch Mm00452152_m1 Mm.298728 nischarin Nlrp3 Mm00840904_m1 Mm.54174 NLR family, pyrin domain containing 3 Nox4 Mm00479246_m1 Mm.31748 NADPH oxidase 4 Npy Mm03048253_m1 Mm.154796 neuropeptide Y Osmr Mm01307326_m1 Mm.10760 oncostatin M receptor Park7 Mm00498538_m1 Mm.277349 Parkinson disease (autosomal recessive, early onset) 7 Pcca Mm00454899_m1 Mm.23876 propionyl-Coenzyme A carboxylase, alpha polypeptide Pccb Mm00452663_m1 Mm.335385 propionyl Coenzyme A carboxylase, beta polypeptide Pdcd1lg2 Mm00451734_m1 Mm.116737 programmed cell death 1 ligand 2 Pdgfrb Mm00435546_m1 Mm.4146 platelet derived growth factor receptor, beta polypeptide Pdha1 Mm00468675_m1 Mm.34775 pyruvate dehydrogenase E1 alpha 1 Pik3c2g Mm00440781_m1 Mm.333471 phosphatidylinositol 3-kinase, C2 domain containing, gamma polypeptide Pik3cg Mm00445038_m1 Mm.101369 phosphoinositide-3-kinase, catalytic, gamma polypeptide Pik3r5 Mm00805206_m1 Mm.244960 phosphoinositide-3-kinase, regulatory subunit 5, p101 Pipox Mm00477190_m1 Mm.8543 pipecolic acid oxidase Pla2g4a Mm00447040_m1 Mm.4186 phospholipase A2, group IVA (cytosolic, calcium-dependent) Pla2g7 Mm00479105_m1 Mm.9277 phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma) Pla2r1 Mm00476896_m1 Mm.5092 phospholipase A2 receptor 1 Plau Mm01274460_g1 Mm.4183 plasminogen activator, urokinase Page 104 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 21 Plcd4 Mm00455768_m1 Mm.290731 phospholipase C, delta 4 Plce1 Mm00457691_m1 Mm.34031 phospholipase C, epsilon 1 Pld4 Mm00626861_m1 Mm.203915 phospholipase D family, member 4 Plekha2 Mm00504233_m1 Mm.261122 pleckstrin homology domain-containing, family A (phosphoinositide binding specific) member 2 Pnmt Mm00476993_m1 Mm.57030 phenylethanolamine-N-methyltransferase Por Mm00435876_m1 Mm.3863 P450 (cytochrome) oxidoreductase Ppara Mm00440939_m1 Mm.212789 peroxisome proliferator activated receptor alpha Ppm1l Mm00618786_m1 Mm.40577 protein phosphatase 1 (formerly 2C)-like Prdx3 Mm00545848_m1 Mm.29821 peroxiredoxin 3 Prdx5 Mm00465365_m1 Mm.279782 peroxiredoxin 5 Prkcb Mm00435749_m1 Mm.207496 protein kinase C, beta Prkcz Mm00776345_g1 Mm.28561 protein kinase C, zeta Pros1 Mm01343426_m1 Mm.127156 protein S (alpha) Prtn3 Mm00478323_m1 Mm.2364 proteinase 3 Psmb8 Mm01278979_m1 Mm.180191 proteasome (prosome, macropain) subunit, beta type 8 (large multifunctional peptidase 7) Psmb9 Mm00479004_m1 Mm.390983 proteasome (prosome, macropain) subunit, beta type 9 (large multifunctional peptidase 2) Ptgs2 Mm00478374_m1 Mm.292547 prostaglandin-endoperoxide synthase 2 Ptpn22 Mm00501246_m1 Mm.395 protein tyrosine phosphatase, non-receptor type 22 (lymphoid) Ptpn6 Mm00469153_m1 Mm.271799 protein tyrosine phosphatase, non-receptor type 6 Ptprc Mm01293577_m1 Mm.391573 protein tyrosine phosphatase, receptor type, C Ptx3 Mm00477268_m1 Mm.276776 pentraxin related gene Pycard Mm00445747_g1 Mm.24163 PYD and CARD domain containing Rac2 Mm00485472_m1 Mm.1972 RAS-related C3 botulinum substrate 2 Rad23a Mm00436249_g1 Mm.255539 RAD23a homolog (S. cerevisiae) Rdh14 Mm00502743_m1 Mm.119343 retinol dehydrogenase 14 (all-trans and 9-cis) Rela Mm00501346_m1 Mm.249966 v-rel reticuloendotheliosis viral oncogene homolog A (avian) Relb Mm00485664_m1 Mm.1741 avian reticuloendotheliosis viral (v-rel) oncogene related B Rnase1 Mm00726747_s1 Mm.235538 ribonuclease, RNase A family, 1 (pancreatic) Robo1 Mm00803879_m1 Mm.310772 roundabout homolog 1 (Drosophila) S100a8 Mm00496696_g1 Mm.21567 S100 calcium binding protein A8 (calgranulin A) Saa1 Mm00656927_g1 Mm.148800 serum amyloid A 1 Sardh Mm00454657_m1 Mm.278467 sarcosine dehydrogenase Scd1 Mm00772290_m1 Mm.267377 stearoyl-Coenzyme A desaturase 1 Sdha Mm01352366_m1 Mm.158231 succinate dehydrogenase complex, subunit A, flavoprotein (Fp) Serpina1a Mm02748447_g1 Mm.439692 serine (or cysteine) peptidase inhibitor, clade A, member 1A Serpina1c; Serpina1a Mm04207709_gH Mm.439692 serine (or cysteine) peptidase inhibitor, clade A, member 1C Serpina1d Mm00842094_mH Mm.439695 serine (or cysteine) peptidase inhibitor, clade A, member 1D Serpina1d; Serpina1b Mm04207706_gH Mm.439692 serine (or cysteine) preptidase inhibitor, clade A, member 1B Serpina1e Mm00833655_m1 Mm.312593 serine (or cysteine) peptidase inhibitor, clade A, member 1E Serpina6 Mm00432327_m1 Mm.290079 serine (or cysteine) peptidase inhibitor, clade A, member 6 Serpine1 Mm00435860_m1 Mm.250422 serine (or cysteine) peptidase inhibitor, clade E, member 1 Serping1 Mm00437834_m1 Mm.38888 serine (or cysteine) peptidase inhibitor, clade G, member 1 Sh3bp2 Mm00449397_m1 Mm.5012 SH3-domain binding protein 2 Sigirr Mm01275624_g1 Mm.38017 single immunoglobulin and toll-interleukin 1 receptor (TIR) domain Slc2a4 Mm01245502_m1 Mm.10661 solute carrier family 2 (facilitated glucose transporter), member 4 Page 105 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 22 Socs3 Mm00545913_s1 Mm.3468 suppressor of cytokine signaling 3 Sod2 Mm01313000_m1 Mm.290876 superoxide dismutase 2, mitochondrial Sod3 Mm01213380_s1 Mm.2407 superoxide dismutase 3, extracellular Sord Mm00455377_g1 Mm.371580 sorbitol dehydrogenase Spi1 Mm00488142_m1 Mm.1302 spleen focus forming virus (SFFV) proviral integration oncogene Spn Mm01164549_m1 Mm.283714 sialophorin Spp1 Mm00436767_m1 Mm.288474 secreted phosphoprotein 1 Srr Mm01246014_m1 Mm.131443 serine racemase Stat1 Mm00439531_m1 Mm.277406 signal transducer and activator of transcription 1 Stat3 Mm01219775_m1 Mm.249934 signal transducer and activator of transcription 3 Stat4 Mm00448890_m1 Mm.1550 signal transducer and activator of transcription 4 Suox Mm00620388_g1 Mm.23352 sulfite oxidase Surf1 Mm00489041_g1 Mm.347512 surfeit gene 1 Syk Mm01333032_m1 Mm.375031 spleen tyrosine kinase Tap1 Mm00443188_m1 Mm.482076 transporter 1, ATP-binding cassette, sub-family B (MDR/TAP) Tap2 Mm01277033_m1 Mm.14814 transporter 2, ATP-binding cassette, sub-family B (MDR/TAP) Tapbp Mm00493417_m1 Mm.154457 TAP binding protein Tgfb1 Mm01178820_m1 Mm.248380 transforming growth factor, beta 1 Tgfbr1 Mm00436964_m1 Mm.197552 transforming growth factor, beta receptor I Thbs2 Mm01279240_m1 Mm.26688 thrombospondin 2 Timp3 Mm00441826_m1 Mm.4871 tissue inhibitor of metalloproteinase 3 Tlr1 Mm00446095_m1 Mm.273024 toll-like receptor 1 Tlr13 Mm01233819_m1 Mm.336203 toll-like receptor 13 Tlr2 Mm00442346_m1 Mm.87596 toll-like receptor 2 Tlr4 Mm00445273_m1 Mm.38049 toll-like receptor 4 Tlr7 Mm00446590_m1 Mm.23979 toll-like receptor 7 Tlr9 Mm00446193_m1 Mm.44889 toll-like receptor 9 Tmem173 Mm01158117_m1 Mm.45995 transmembrane protein 173 Tnf Mm00443258_m1 Mm.1293 tumor necrosis factor Tnfaip3 Mm00437121_m1 Mm.116683 tumor necrosis factor, alpha-induced protein 3 Tnfrsf12a Mm01302476_g1 Mm.28518 tumor necrosis factor receptor superfamily, member 12a Tnfrsf13b Mm00840182_m1 Mm.265915 tumor necrosis factor receptor superfamily, member 13b Tnfrsf13c Mm00840578_g1 Mm.240047 tumor necrosis factor receptor superfamily, member 13c Tnfrsf17 Mm00495683_m1 Mm.12935 tumor necrosis factor receptor superfamily, member 17 Tnfsf12 Mm02583406_s1 Mm.8983 tumor necrosis factor (ligand) superfamily, member 12 Tnfsf13 Mm03809849_s1 Mm.8983 tumor necrosis factor (ligand) superfamily, member 13 Tnfsf13b Mm00446347_m1 Mm.28835 tumor necrosis factor (ligand) superfamily, member 13b Tnfsf4 Mm00437214_m1 Mm.4994 tumor necrosis factor (ligand) superfamily, member 4 Tnip1 Mm01288484_m1 Mm.259671 TNFAIP3 interacting protein 1 Tnip2 Mm00460482_m1 Mm.28615 TNFAIP3 interacting protein 2 Tnip3 Mm01181626_m1 Mm.117558 TNFAIP3 interacting protein 3 Traf1 Mm00493827_m1 Mm.239514 TNF receptor-associated factor 1 Traf2 Mm00801978_m1 Mm.3399 TNF receptor-associated factor 2 Trak1 Mm00613053_m1 Mm.491112 trafficking protein, kinesin binding 1 Trem2 Mm04209424_g1 Mm.261623 triggering receptor expressed on myeloid cells 2 Tst Mm01195231_m1 Mm.15312 thiosulfate sulfurtransferase, mitochondrial Page 106 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 23 Txnrd2 Mm00496766_m1 Mm.390906 thioredoxin reductase 2 Tyrobp Mm00449152_m1 Mm.46301 TYRO protein tyrosine kinase binding protein Ugt2b34 Mm00655373_m1 Mm.281844 UDP glucuronosyltransferase 2 family, polypeptide B34 Ugt2b35 Mm00655596_m1 Mm.312095 UDP glucuronosyltransferase 2 family, polypeptide B35 Umod Mm00447649_m1 Mm.10826 uromodulin Uqcrc1 Mm00445911_m1 Mm.335460 ubiquinol-cytochrome c reductase core protein 1 Uqcrc2 Mm00445961_m1 Mm.334206 ubiquinol cytochrome c reductase core protein 2 Uqcrfs1 Mm00481849_m1 Mm.181933 ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 Vamp3 Mm01268442_g1 Mm.273930 vesicle-associated membrane protein 3 Vav1 Mm01232047_m1 Mm.248172 vav 1 oncogene Vcam1 Mm01320970_m1 Mm.440909 vascular cell adhesion molecule 1 Vegfa Mm01281449_m1 Mm.282184 vascular endothelial growth factor A Vegfc Mm00437310_m1 Mm.1402 vascular endothelial growth factor C Was Mm00494167_m1 Mm.4735 Wiskott-Aldrich syndrome homolog (human) Zbtb46 Mm00511327_m1 Mm.486504 zinc finger and BTB domain containing 46 Zfp36 Mm00457144_m1 Mm.389856 zinc finger protein 36 Page 107 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 24 Table S2.- List of genes induced by C4BP(-) treatment (FC > 1.8) Gene Symbol Log2(FC) C4BP(β-) Assay ID UniGene ID Gene name Chga 9,2 Mm00514341_m1 Mm.4137 chromogranin A Pnmt 9,1 Mm00476993_m1 Mm.57030 phenylethanolamine-N-methyltransferase Adh7 4,1 Mm00507750_m1 Mm.8473 alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide Robo1 1,1 Mm00803879_m1 Mm.310772 roundabout homolog 1 (Drosophila) Serpina1a 1,0 Mm02748447_g1 Mm.439692 serine (or cysteine) peptidase inhibitor, clade A, member 1A Adh1 1,0 Mm00507711_m1 Mm.2409 alcohol dehydrogenase 1 (class I) Serpina6 1,0 Mm00432327_m1 Mm.290079 serine (or cysteine) peptidase inhibitor, clade A, member 6 Igfbp6 0,9 Mm00599696_m1 Mm.358609 insulin-like growth factor binding protein 6 Ccr6 -0,9 Mm99999114_s1 Mm.8007 chemokine (C-C motif) receptor 6 Cst7 -0,9 Mm00438349_m1 Mm.12965 cystatin F (leukocystatin) Psmb8 -0,9 Mm01278979_m1 Mm.180191 proteasome (prosome, macropain) subunit, beta type 8 (large multifunctional peptidase 7) Cd22 -0,9 Mm00515432_m1 Mm.260994 CD22 antigen Havcr1 -0,9 Mm00506686_m1 Mm.17771 hepatitis A virus cellular receptor 1 Blk -0,9 Mm00432077_m1 Mm.3962 B lymphoid kinase Cd3d -0,9 Mm00442746_m1 Mm.4527 CD3 antigen, delta polypeptide Lat -1,0 Mm00456761_m1 Mm.10280 linker for activation of T cells Tnfrsf13c -1,1 Mm00840578_g1 Mm.240047 tumor necrosis factor receptor superfamily, member 13c Irf7 -1,2 Mm00516793_g1 Mm.3233 interferon regulatory factor 7 Prtn3 -1,3 Mm00478323_m1 Mm.2364 proteinase 3 Ms4a1 -1,4 Mm00545909_m1 Mm.4046 membrane-spanning 4-domains, subfamily A, member 1 S100a8 -1,6 Mm00496696_g1 Mm.21567 S100 calcium binding protein A8 (calgranulin A) Cd19 -1,6 Mm00515420_m1 Mm.4360 CD19 antigen Il12a -2,8 Mm00434165_m1 Mm.103783 interleukin 12a Saa1 -3,4 Mm00656927_g1 Mm.148800 serum amyloid A 1 FC: Fold change Page 108 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 25 Table S3.- List of genes induced by CYP treatment (FC > 1.8) Gene Symbol Log2(FC) CYP Assay ID UniGene ID Gene name Serpina6 2,5 Mm00432327_m1 Mm.290079 serine (or cysteine) peptidase inhibitor, clade A, member 6 Serpina1d 2,1 Mm00842094_mH Mm.439695 serine (or cysteine) peptidase inhibitor, clade A, member 1D Npy 1,7 Mm03048253_m1 Mm.154796 neuropeptide Y Igfbp1 1,6 Mm00515154_m1 Mm.21300 insulin-like growth factor binding protein 1 C8a 1,5 Mm00521627_m1 Mm.197638 complement component 8, alpha polypeptide Serpina1a 1,5 Mm02748447_g1 Mm.439692 serine (or cysteine) peptidase inhibitor, clade A, member 1A Aldh6a1 1,3 Mm00506227_m1 Mm.247510 aldehyde dehydrogenase family 6, subfamily A1 Ndrg2 1,2 Mm00443481_g1 Mm.26722 N-myc downstream regulated gene 2 Ace 1,2 Mm00802048_m1 Mm.754 angiotensin I converting enzyme (peptidyl-dipeptidase A) 1 Bckdhb 1,1 Mm01177077_m1 Mm.12819 branched chain ketoacid dehydrogenase E1, beta polypeptide Slc2a4 1,1 Mm01245502_m1 Mm.10661 solute carrier family 2 (facilitated glucose transporter), member 4 Aldh4a1 1,1 Mm00615268_m1 Mm.273571 aldehyde dehydrogenase 4 family, member A1 Plcd4 1,1 Mm00455768_m1 Mm.290731 phospholipase C, delta 4 Por 1,1 Mm00435876_m1 Mm.3863 P450 (cytochrome) oxidoreductase Fmo1 1,1 Mm00515795_m1 Mm.976 flavin containing monooxygenase 1 Pik3c2g 1,1 Mm00440781_m1 Mm.333471 phosphatidylinositol 3-kinase, C2 domain containing, gamma polypeptide Klkb1 1,0 Mm00434658_m1 Mm.482691 kallikrein B, plasma 1 Cbs 1,0 Mm00460654_m1 Mm.206417 cystathionine beta-synthase Sod2 1,0 Mm01313000_m1 Mm.290876 superoxide dismutase 2, mitochondrial Ugt2b34 1,0 Mm00655373_m1 Mm.281844 UDP glucuronosyltransferase 2 family, polypeptide B34 Fmo4 1,0 Mm00467393_m1 Mm.155164 flavin containing monooxygenase 4 Nox4 1,0 Mm00479246_m1 Mm.31748 NADPH oxidase 4 Ahcyl2 1,0 Mm00619649_m1 Mm.210899 S-adenosylhomocysteine hydrolase-like 2 Scd1 1,0 Mm00772290_m1 Mm.267377 stearoyl-Coenzyme A desaturase 1 Prkcz 1,0 Mm00776345_g1 Mm.28561 protein kinase C, zeta Cat 1,0 Mm00437992_m1 Mm.4215 catalase Adhfe1 1,0 Mm00613830_m1 Mm.28514 alcohol dehydrogenase, iron containing, 1 Serpina1d; Serpina1b 1,0 Mm04207706_gH Mm.439692 serine (or cysteine) preptidase inhibitor, clade A, member 1B Idh3b 0,9 Mm00504589_m1 Mm.29590 isocitrate dehydrogenase 3 (NAD+) beta Sardh 0,9 Mm00454657_m1 Mm.278467 sarcosine dehydrogenase Sord 0,9 Mm00455377_g1 Mm.371580 sorbitol dehydrogenase Id1 0,9 Mm00775963_g1 Mm.444 inhibitor of DNA binding 1 Aldh9a1 0,9 Mm00480240_m1 Mm.292539 aldehyde dehydrogenase 9, subfamily A1 Sdha 0,9 Mm01352366_m1 Mm.158231 succinate dehydrogenase complex, subunit A, flavoprotein (Fp) Bckdha 0,9 Mm00476112_m1 Mm.25848 branched chain ketoacid dehydrogenase E1, alpha polypeptide Suox 0,9 Mm00620388_g1 Mm.23352 sulfite oxidase Tst 0,9 Mm01195231_m1 Mm.15312 thiosulfate sulfurtransferase, mitochondrial Pcca 0,9 Mm00454899_m1 Mm.23876 propionyl-Coenzyme A carboxylase, alpha polypeptide Idh3g 0,9 Mm00599686_g1 Mm.14825 isocitrate dehydrogenase 3 (NAD+), gamma Hnf4a 0,9 Mm01247712_m1 Mm.202383 hepatic nuclear factor 4, alpha Ndufa10 0,9 Mm00600325_m1 Mm.248778 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10 Ppm1l 0,9 Mm00618786_m1 Mm.40577 protein phosphatase 1 (formerly 2C)-like Prdx3 0,9 Mm00545848_m1 Mm.29821 peroxiredoxin 3 Got1 0,9 Mm01195792_g1 Mm.19039 glutamic-oxaloacetic transaminase 1, soluble Ndufaf1 0,9 Mm00452828_m1 Mm.5390 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, assembly factor 1 Gss 0,9 Mm00515065_m1 Mm.252316 glutathione synthetase Auh 0,9 Mm00479363_m1 Mm.252034 AU RNA binding protein/enoyl-coenzyme A hydratase Ndufs7 0,9 Mm01144210_m1 Mm.28712 NADH dehydrogenase (ubiquinone) Fe-S protein 7 Atp5f1 0,9 Mm01296543_g1 Mm.251152 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit B1 Timp3 0,8 Mm00441826_m1 Mm.4871 tissue inhibitor of metalloproteinase 3 Aldh2 0,8 Mm00477463_m1 Mm.284446 aldehyde dehydrogenase 2, mitochondrial Stat3 -0,9 Mm01219775_m1 Mm.249934 signal transducer and activator of transcription 3 Irf1 -0,9 Mm01288580_m1 Mm.105218 interferon regulatory factor 1 B2m -0,9 Mm00437762_m1 Mm.163 beta-2 microglobulin Cx3cl1 -0,9 Mm00436454_m1 Mm.103711 chemokine (C-X3-C motif) ligand 1 Page 109 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 26 Ctgf -0,9 Mm01192932_g1 Mm.390287 connective tissue growth factor Hmox1 -0,9 Mm00516005_m1 Mm.276389 heme oxygenase 1 F3 -0,9 Mm00438853_m1 Mm.273188 coagulation factor III Tnfaip3 -0,9 Mm00437121_m1 Mm.116683 tumor necrosis factor, alpha-induced protein 3 Icam1 -0,9 Mm00516023_m1 Mm.435508 intercellular adhesion molecule 1 Tgfb1 -1,0 Mm01178820_m1 Mm.248380 transforming growth factor, beta 1 Sh3bp2 -1,0 Mm00449397_m1 Mm.5012 SH3-domain binding protein 2 Lyn -1,0 Mm01217488_m1 Mm.317331 Yamaguchi sarcoma viral (v-yes-1) oncogene homolog Traf1 -1,0 Mm00493827_m1 Mm.239514 TNF receptor-associated factor 1 Apoe -1,0 Mm01307193_g1 Mm.305152 apolipoprotein E Tnfsf4 -1,0 Mm00437214_m1 Mm.4994 tumor necrosis factor (ligand) superfamily, member 4 Cebpb -1,0 Mm00843434_s1 Mm.439656 CCAAT/enhancer binding protein (C/EBP), beta Irf9 -1,0 Mm00492679_m1 Mm.2032 interferon regulatory factor 9 Inpp5d -1,0 Mm00494987_m1 Mm.15105 inositol polyphosphate-5-phosphatase D Igfbp6 -1,0 Mm00599696_m1 Mm.358609 insulin-like growth factor binding protein 6 Cfh -1,0 Mm01299248_m1 Mm.8655 complement component factor h Arpc1b -1,0 Mm00834862_m1 Mm.30010 actin related protein 2/3 complex, subunit 1B C1qc -1,0 Mm00776126_m1 Mm.439732 complement component 1, q subcomponent, C chain C4bp -1,0 Mm00432150_m1 Mm.306720 complement component 4 binding protein Cxcr4 -1,0 Mm01996749_s1 Mm.1401 chemokine (C-X-C motif) receptor 4 C1ra -1,1 Mm04206253_g1 Mm.333375 complement component 1, r subcomponent A Fga -1,1 Mm00802584_m1 Mm.88793 fibrinogen alpha chain Csf1 -1,1 Mm00432686_m1 Mm.795 colony stimulating factor 1 (macrophage) C1s1 -1,1 Mm00663210_mH Mm.219527 complement component 1, s subcomponent 1 Ikbke -1,1 Mm00444862_m1 Mm.386783 inhibitor of kappaB kinase epsilon Map3k8 -1,2 Mm00432637_m1 Mm.3275 mitogen-activated protein kinase kinase kinase 8 Pycard -1,2 Mm00445747_g1 Mm.24163 PYD and CARD domain containing Tlr4 -1,2 Mm00445273_m1 Mm.38049 toll-like receptor 4 Ccr1 -1,2 Mm00438260_s1 Mm.274927 chemokine (C-C motif) receptor 1 Cxcl9 -1,2 Mm00434946_m1 Mm.766 chemokine (C-X-C motif) ligand 9 Thbs2 -1,2 Mm01279240_m1 Mm.26688 thrombospondin 2 Psmb9 -1,2 Mm00479004_m1 Mm.390983 proteasome (prosome, macropain) subunit, beta type 9 (large multifunctional peptidase 2) Il2rg -1,3 Mm00442885_m1 Mm.2923 interleukin 2 receptor, gamma chain Ncf1 -1,3 Mm00447921_m1 Mm.425296 neutrophil cytosolic factor 1 Relb -1,3 Mm00485664_m1 Mm.1741 avian reticuloendotheliosis viral (v-rel) oncogene related B Cxcl10 -1,3 Mm00445235_m1 Mm.877 chemokine (C-X-C motif) ligand 10 Mpo -1,3 Mm01298424_m1 Mm.4668 myeloperoxidase Fgg -1,3 Mm00513575_m1 Mm.16422 fibrinogen gamma chain Cd1d1 -1,4 Mm00783541_s1 Mm.1894 CD1d1 antigen Ifng -1,4 Mm01168134_m1 Mm.240327 interferon gamma Fgb -1,4 Mm00805336_m1 Mm.30063 fibrinogen beta chain S100a8 -1,4 Mm00496696_g1 Mm.21567 S100 calcium binding protein A8 (calgranulin A) Serping1 -1,4 Mm00437834_m1 Mm.38888 serine (or cysteine) peptidase inhibitor, clade G, member 1 Nfkbie -1,4 Mm01269649_m1 Mm.57043 nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, epsilon Tlr7 -1,4 Mm00446590_m1 Mm.23979 toll-like receptor 7 Ptpn22 -1,4 Mm00501246_m1 Mm.395 protein tyrosine phosphatase, non-receptor type 22 (lymphoid) Tlr1 -1,4 Mm00446095_m1 Mm.273024 toll-like receptor 1 Il33 -1,4 Mm00505403_m1 Mm.182359 interleukin 33 Nlrp3 -1,4 Mm00840904_m1 Mm.54174 NLR family, pyrin domain containing 3 Tnf -1,4 Mm00443258_m1 Mm.1293 tumor necrosis factor Mmp9 -1,5 Mm00442991_m1 Mm.4406 matrix metallopeptidase 9 Tap1 -1,5 Mm00443188_m1 Mm.482076 transporter 1, ATP-binding cassette, sub-family B (MDR/TAP) Tnip3 -1,5 Mm01181626_m1 Mm.117558 TNFAIP3 interacting protein 3 Casp1 -1,5 Mm00438023_m1 Mm.1051 caspase 1 Prkcb -1,5 Mm00435749_m1 Mm.207496 protein kinase C, beta Il12b -1,5 Mm00434174_m1 Mm.239707 interleukin 12b Il27 -1,5 Mm00461162_m1 Mm.222632 interleukin 27 Ccl5 -1,5 Mm01302427_m1 Mm.284248 chemokine (C-C motif) ligand 5 Spi1 -1,5 Mm00488142_m1 Mm.1302 spleen focus forming virus (SFFV) proviral integration oncogene Chga -1,5 Mm00514341_m1 Mm.4137 chromogranin A Page 110 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 27 Csf2rb -1,5 Mm00655745_m1 Mm.235324 colony stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage) Tyrobp -1,5 Mm00449152_m1 Mm.46301 TYRO protein tyrosine kinase binding protein Itga4 -1,5 Mm01277951_m1 Mm.31903 integrin alpha 4 Il27ra -1,6 Mm00497259_m1 Mm.38386 interleukin 27 receptor, alpha Cst7 -1,6 Mm00438349_m1 Mm.12965 cystatin F (leukocystatin) Spn -1,6 Mm01164549_m1 Mm.283714 sialophorin Ikzf1 -1,6 Mm01187882_m1 Mm.103545 IKAROS family zinc finger 1 Pik3cg -1,6 Mm00445038_m1 Mm.101369 phosphoinositide-3-kinase, catalytic, gamma polypeptide Lck -1,6 Mm00802897_m1 Mm.293753 lymphocyte protein tyrosine kinase Stat4 -1,6 Mm00448890_m1 Mm.1550 signal transducer and activator of transcription 4 Tlr9 -1,6 Mm00446193_m1 Mm.44889 toll-like receptor 9 Tnfsf13b -1,6 Mm00446347_m1 Mm.28835 tumor necrosis factor (ligand) superfamily, member 13b Psmb8 -1,6 Mm01278979_m1 Mm.180191 proteasome (prosome, macropain) subunit, beta type 8 (large multifunctional peptidase 7) Trem2 -1,6 Mm04209424_g1 Mm.261623 triggering receptor expressed on myeloid cells 2 Cx3cr1 -1,6 Mm02620111_s1 Mm.44065 chemokine (C-X3-C motif) receptor 1 Was -1,6 Mm00494167_m1 Mm.4735 Wiskott-Aldrich syndrome homolog (human) Il27ra -1,6 Mm00497259_m1 Mm.38386 interleukin 27 receptor, alpha Rac2 -1,6 Mm00485472_m1 Mm.1972 RAS-related C3 botulinum substrate 2 Cd83 -1,6 Mm00486868_m1 Mm.57175 CD83 antigen Vav1 -1,6 Mm01232047_m1 Mm.248172 vav 1 oncogene Btk -1,7 Mm00442712_m1 Mm.4475 Bruton agammaglobulinemia tyrosine kinase Blk -1,7 Mm00432077_m1 Mm.3962 B lymphoid kinase Ccr7 -1,7 Mm01301785_m1 Mm.2932 chemokine (C-C motif) receptor 7 Pdcd1lg2 -1,7 Mm00451734_m1 Mm.116737 programmed cell death 1 ligand 2 Cd22 -1,7 Mm00515432_m1 Mm.260994 CD22 antigen Tmem173 -1,7 Mm01158117_m1 Mm.45995 transmembrane protein 173 Cd86 -1,7 Mm00444543_m1 Mm.1452 CD86 antigen Itgb2 -1,7 Mm00434513_m1 Mm.1137 integrin beta 2 Hck -1,7 Mm01241463_m1 Mm.715 hemopoietic cell kinase Tnfrsf12a -1,7 Mm01302476_g1 Mm.28518 tumor necrosis factor receptor superfamily, member 12a Clec7a -1,7 Mm01183349_m1 Mm.239516 C-type lectin domain family 7, member a Cd79b -1,7 Mm00434143_m1 Mm.2987 CD79B antigen Icos -1,7 Mm00497600_m1 Mm.42044 inducible T cell co-stimulator Tlr13 -1,8 Mm01233819_m1 Mm.336203 toll-like receptor 13 Fasl -1,8 Mm00438864_m1 Mm.3355 Fas ligand (TNF superfamily, member 6) Fcer1g -1,8 Mm02343757_m1 Mm.22673 Fc receptor, IgE, high affinity I, gamma polypeptide Aim2 -1,8 Mm01295719_m1 Mm.131453 absent in melanoma 2 Pld4 -1,8 Mm00626861_m1 Mm.203915 phospholipase D family, member 4 C1qa -1,8 Mm00432142_m1 Mm.439957 complement component 1, q subcomponent, alpha polypeptide Cd276 -1,8 Mm00506020_m1 Mm.5356 CD276 antigen Tnfrsf13b -1,8 Mm00840182_m1 Mm.265915 tumor necrosis factor receptor superfamily, member 13b Osmr -1,8 Mm01307326_m1 Mm.10760 oncostatin M receptor Cd74 -1,8 Mm00658576_m1 Mm.439737 CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated) Prtn3 -1,8 Mm00478323_m1 Mm.2364 proteinase 3 Syk -1,8 Mm01333032_m1 Mm.375031 spleen tyrosine kinase Cd48 -1,9 Mm00455932_m1 Mm.1738 CD48 antigen Il10ra -1,9 Mm00434151_m1 Mm.379327 interleukin 10 receptor, alpha Cxcl1 -1,9 Mm04207460_m1 Mm.21013 chemokine (C-X-C motif) ligand 1 Irf7 -1,9 Mm00516793_g1 Mm.3233 interferon regulatory factor 7 Clec10a -1,9 Mm00546125_g1 Mm.252405 C-type lectin domain family 10, member A C3ar1 -1,9 Mm02620006_s1 Mm.2408 complement component 3a receptor 1 C1qb -1,9 Mm01179619_m1 Mm.2570 complement component 1, q subcomponent, beta polypeptide Fcgr3 -1,9 Mm00438882_m1 Mm.22119 Fc receptor, IgG, low affinity III Cd3g -1,9 Mm00438095_m1 Mm.335106 CD3 antigen, gamma polypeptide Clu -1,9 Mm01197002_m1 Mm.200608 clusterin Ltb -2,0 Mm00434774_g1 Mm.1715 lymphotoxin B Lcp2 -2,0 Mm01187570_m1 Mm.265350 lymphocyte cytosolic protein 2 Ifi202b -2,0 Mm00839397_m1 Mm.218770 interferon activated gene 202B Lat -2,0 Mm00456761_m1 Mm.10280 linker for activation of T cells Vcam1 -2,0 Mm01320970_m1 Mm.440909 vascular cell adhesion molecule 1 Page 111 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 28 Il1r2 -2,0 Mm00439629_m1 Mm.1349 interleukin 1 receptor, type II Pik3r5 -2,0 Mm00805206_m1 Mm.244960 phosphoinositide-3-kinase, regulatory subunit 5, p101 Ptprc -2,0 Mm01293577_m1 Mm.391573 protein tyrosine phosphatase, receptor type, C Spp1 -2,0 Mm00436767_m1 Mm.288474 secreted phosphoprotein 1 Ccr2 -2,0 Mm00438270_m1 Mm.6272 chemokine (C-C motif) receptor 2 Col1a2 -2,1 Mm00483888_m1 Mm.277792 collagen, type I, alpha 2 Fn1 -2,1 Mm01256744_m1 Mm.193099 fibronectin 1 Cd80 -2,1 Mm00711660_m1 Mm.89474 CD80 antigen Foxp3 -2,1 Mm00475162_m1 Mm.182291 forkhead box P3 Cd40lg -2,1 Mm00441911_m1 Mm.4861 CD40 ligand Clec4n -2,2 Mm00490934_m1 Mm.271782 C-type lectin domain family 4, member n Pnmt -2,2 Mm00476993_m1 Mm.57030 phenylethanolamine-N-methyltransferase Ctla4 -2,2 Mm00486849_m1 Mm.390 cytotoxic T-lymphocyte-associated protein 4 Msr1 -2,2 Mm00446214_m1 Mm.239291 macrophage scavenger receptor 1 Il1b -2,2 Mm00434228_m1 Mm.222830 interleukin 1 beta Cd28 -2,2 Mm00483137_m1 Mm.255003 CD28 antigen Ccr6 -2,2 Mm99999114_s1 Mm.8007 chemokine (C-C motif) receptor 6 Cd3d -2,3 Mm00442746_m1 Mm.4527 CD3 antigen, delta polypeptide Il10 -2,3 Mm00439614_m1 Mm.874 interleukin 10 Gpnmb -2,3 Mm01328587_m1 Mm.302602 glycoprotein (transmembrane) nmb Serpine1 -2,3 Mm00435860_m1 Mm.250422 serine (or cysteine) peptidase inhibitor, clade E, member 1 Ctss -2,3 Mm01255859_m1 Mm.3619 cathepsin S Ccl9 -2,3 Mm00441260_m1 Mm.416125 chemokine (C-C motif) ligand 9 Cd19 -2,3 Mm00515420_m1 Mm.4360 CD19 antigen Cd300lb -2,3 Mm01701741_m1 Mm.185355 CD300 antigen like family member B Fcgr4 -2,3 Mm00519988_m1 Mm.251254 Fc receptor, IgG, low affinity IV Cd5 -2,4 Mm00432417_m1 Mm.779 CD5 antigen Tnfrsf17 -2,4 Mm00495683_m1 Mm.12935 tumor necrosis factor receptor superfamily, member 17 Tnfrsf13c -2,4 Mm00840578_g1 Mm.240047 tumor necrosis factor receptor superfamily, member 13c Irf4 -2,4 Mm00516431_m1 Mm.4677 interferon regulatory factor 4 Fcgr1 -2,4 Mm00438874_m1 Mm.150 Fc receptor, IgG, high affinity I Ccl2 -2,4 Mm00441242_m1 Mm.290320 chemokine (C-C motif) ligand 2 Ms4a1 -2,5 Mm00545909_m1 Mm.4046 membrane-spanning 4-domains, subfamily A, member 1 Havcr1 -2,5 Mm00506686_m1 Mm.17771 hepatitis A virus cellular receptor 1 Cd14 -2,5 Mm00438094_g1 Mm.3460 CD14 antigen Itgam -2,5 Mm00434455_m1 Mm.262106 integrin alpha M Il7r -2,6 Mm00434295_m1 Mm.389 interleukin 7 receptor C3 -2,7 Mm00437838_m1 Mm.19131 complement component 3 Cxcl2 -2,7 Mm00436450_m1 Mm.4979 chemokine (C-X-C motif) ligand 2 Ereg -2,7 Mm00514794_m1 Mm.4791 epiregulin Il17a -2,7 Mm00439618_m1 Mm.5419 interleukin 17A Col3a1 -2,7 Mm01254476_m1 Mm.249555 collagen, type III, alpha 1 Ccr5 -2,7 Mm01963251_s1 Mm.14302 chemokine (C-C motif) receptor 5 Socs3 -2,8 Mm00545913_s1 Mm.3468 suppressor of cytokine signaling 3 C6 -2,8 Mm00489521_m1 Mm.20247 complement component 6 Col1a1 -3,0 Mm00801666_g1 Mm.277735 collagen, type I, alpha 1 Cxcl13 -3,1 Mm04214185_s1 Mm.10116 chemokine (C-X-C motif) ligand 13 Saa1 -4,5 Mm00656927_g1 Mm.148800 serum amyloid A 1 Il1f6 -6,0 Mm00457645_m1 Mm.133095 interleukin 1 family, member 6 FC: Fold change Page 112 of 121 The International Society of Nephrology (http://www.isn-online.org/site/cms) Kidney International 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only Luque et al. (September 2019) KI-12-18-1865 29 Table S4.- Relevant enriched biological functions (IPA®) associated with differentially expressed renal genes from C4BP(β-)-treated NZBW F1 mice. Bio-function # genes in dataset Overlap p-value Activation z-score* Quantity of leukocytes 14 2,91x10-11 -2,836 Activation of leukocytes 10 6,03x10-10 -1,455 Quantity of mononuclear leukocytes 12 6,06x10-10 -2,541 Quantity of lymphocytes 11 6,87x10-9 -2,839 Leukocyte migration 9 8,12x10-7 -1,966 Activation of lymphocytes 6 5,71x10-6 -1,368 Quantity of B lymphocytes 6 1,41x10-5 -2,223 Cell viability of lymphocytes 4 4,15x10-5 -1,969 Cell movement of phagocytes 5 3,53x10-4 -2,211 Cell movement of myeloid cells 5 3,59x10-4 -2,195 Cellular infiltration by leukocytes 4 1,90x10-3 -1,972 Inflammation of organ 5 7,56x10-3 0,753 *Activation z-score is calculated by the IPA software and predicts whether a specific disease or bio- function is increased (positive z-score) or decreased (negative z-score) based on the experimental dataset. In bold are significant Bio-functions with z-score >2 or < -2. 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