L. Mas

Establishing the molecular pathways involved in chronic allograft nephropathy for testing new noninvasive diagnostic markers.

Background. Chronic allograft nephropathy (CAN) is a cause of graft loss. The multistage processes that result in CAN are poorly understood. Noninvasive assays for detecting allograft dysfunction and predicting long-term outcomes are a priority in transplantation (Tx). Methods. Renal tissue from kidney transplant patients (KTP) with CAN (n=11) and normal kidneys (NK; n=7) were studied using microarrays. Markers resulting from the microarray analysis (transforming growth factor [TGF]-β, epidermal growth factor receptor [EGFR], angiotensinogen [AGT]) were tested in urine (Ur) and peripheral blood (PB) samples from the CAN patients (collected at the biopsy time) using reverse-transcriptase real-time polymerase chain reaction. Ur and PB samples from long-term KTP with stable renal function (SRF; n=20) were used as control. Results. Assuming unequal variances between CAN and NK, using a false discovery rate of 0.005, and running 1,000 of all possible permutations, 728 probe sets were differentially expressed. Genes related to fibrosis and extracellular matrix deposition (i.e., TGF-β, laminin, gamma 2, metalloproteinases-9, and collagen type IX alpha 3) were up-regulated. Genes related to immunoglobulins, B cells, T-cell receptor, nuclear factor of activated T cells, and cytokine and chemokines receptors were also upregulated. EGFR and growth factor receptor activity (FGFR)2 were downregulated in CAN samples. AGT, EGFR, and TGF-β levels were statistical different in urine but not in blood samples of CAN patients when compared to KTP with SRF (P<0.001, P=0.04, and P<0.001, respectively). Conclusions. Genes related to fibrosis, extracellular matrix deposition, and immune response were found up-regulated in CAN. Markers resulting from the microarray analysis were differentially expressed in Ur samples of the CAN patients and in concordance with the microarray profiles.

THU0289 B-cell activating factor gene expression in urinary sample and renal biopsy for monitoring disease activity in lupus nephritis

Objectives To evaluate BLyS as biomarker in disease activity in urinary sample and renal biopsy from patients with LN. Methods Retrospective study. Between June 2009 and October 2013, 32 patients with SLE and LN fullfilling SLE classification criteria of ACR 1997 were included. The renal biopsies were evaluated according to the ISN/RPS classification system. The gene expression levels of BLyS were quantified using Quantitative Real Time PCR (QPCR). The relative quantification method was used for analysis, where Ct was normalized to an endogenous control β2Microglobulina (β2M) (ΔCt BLyS). The data expressed as ΔCt are inversely proportional to gene expression level. The value of BLyS is expressed as median (M) and interquartile range (IQR) for filing a non-normal distribution. Results 26 (81.3%) patients were female with a mean age at diagnosis of 26.9±13 years and 31.9±29 years at the time of renal biopsy. The SLEDAI at the time of biopsy was 10.5 (IQR 0–15.7) and SLICC ≥1 in 13 (32.5%), hypocomplementemia 13/31 (41.9%) and positive DNA in 11/29 (37.9%) patients. Biopsies from patients with proteinuria ≥0.5 and renal failure (RF) (n=23, 71.9%), proteinuria isolated (n=14, 43.8%), LN remission. The value of the BLyS gene expression in renal biopsy was 8.09 (IQR 7.37–9.16) and BLyS in urinary sample was 6.45 (IQR 5.62–7.76).Table 1. BLyS gene expression in urinary sample and renal biopsy according to clinical and histological findings Variables ΔCt BLyS Urinary p ΔCt BLyS Biopsy p SLEDAI =0/ SLEDAI ≥6 7.52 (6.59–11.19)/5.94 (5.52–7.08) 0.04 8.03 (6.90–10.20)/8.15 (7.35–9.10) 0.82 Bx LN in remission/LN active 7.52 (6.59–11.19)/5.94 (5.52–7.08) 0.04 8.03 (6.90–10.20)/8.15 (7.35–9.10) 0.82 MDRD ≥60/ MDRD ≤60 6.76 (6.018.12)/5.60 (5.16–7.19) 0.04 8.03 (6.95–8.95)/8.41 (7.45–13.40) 0.42 Proteinuria ≤0.5/Proteinuria ≥0.5 7.52 (6.59–11.19)/5.94 (5.52–7.08) 0.04 8.03 (6.90–10.20)/8.15 (7.35–9.10) 0.82 Without tubular atrophy in Bx /Tubular atrophy in Bx 7.83 (6.43–11.67)/6.14 (5.51–7.24) 0.03 8.16 (6.44–8.52)/8.09 (7.42–10.55) 0.53Table 2. BLyS gene expression in urinary sample and renal biopsy according to the classification of LN Class I/Normal Bx Class II Bx Class IV Bx Class V/VI Bx P ΔCt BLyS Renal Biopsy 7.56 (6.50–7.56) 8.41 (7.36–10.37) 7.95 (7.37–10.04) 8.28 (7.37–11.80) 0.88 ΔCt BLyS Urinary*** 7.28 (9.95–7.28) 8.41 (7.36–10.37) 5.65 (5.45–6.40) 6.34 (5.74–7.32) 0.003 ***p<0.05 Class I/normal with class IV, Class II with IV, class II with V/VI. Conclusions BLyS detection in urinary samples could be a potential biomarker for predicting lupus nephritis activity. Our data confirm that the BLyS as urinary biomarker is present in patients with active renal disease especially in patients with proliferative glomerulonephritis. Disclosure of Interest None declared

THU0018 Angiotensinogen as a marker of injury in lupus nephritis

Background Lupus Nephritis (LN) is one of the most severe forms of systemic lupus erythematosus (SLE) (1). Angiotensinogen (AGT) gene encodes the only glycoprotein known to be a precursor of the vasopresor angiotensin II (Ang II). Ang II is also a growth factor and a profibrogenic cytokine (2). In kidney transplantation AGT has been founded down expressed in biopsies with chronic allograft dysfunction (3). In LN, AGT deserves evaluation. Objectives To investigate AGT expression in biopsies and urines from LN patients. Methods 32 biopsies/urines paired from 32 LN patients was included. Kidney biopsies were evaluated according to the ISN/RPS classification system. Levels of AGT were evaluated using Quantitative Real Time PCR. Threshold cycle (Ct) scores were averaged for calculations of relative expression values. The Ct scores were normalized against Ct scores by subtracting β2Microglobuline control, or ΔCt=Ct,gene- Ct,B2M. Data expressed as ΔCt are inversely proportional to gene expression level. Nonparametric Mann Whitney test analysis and Anova with Bonferroni test were performed. Results 26 (81.3%) patients were female with a mean age at biopsy time of 31.9±29 years. The SLEDAI at the time of biopsy was 10.5 (IQR 0–15.7) and SLICC ≥1 in 13 (32.5%), hypocomplementemia 13/31 (41.9%) and positive DNA in 11/29 (37.9%) patients. Biopsies from patients with proteinuria ≥0.5 and renal failure (n=23), proteinuria isolated (n=14), LN remission (n=9), renal failure (n=7) and nephrotic syndrome (n=2) were performed. The mean value of ΔCt AGT gene expression in renal biopsy was 4.50 (IQR 3.51 – 5.67) and AGT in urine samples was 13.94 (IQR 11.66 – 17.89).Table 1. AGT gene expression in biopsies and urines samples Class I/ Class II Class IV Class V/VI p Normal Biopsies Biopsies Biopsies Biopsies n=3 n=6 n=12 n=10 p ΔCt AGT Biopsies* 5,57 3,67 5,34 4,35 0,02 (3,60–5,57) (2,19–5,37) (4,75–10,93) (3,45–4,57) ΔCt AGT Urines** 14,11 11,19 16,77 15,06 0,01 (12,44–14,11) (9,53–11,59) (12,88–18,25) (12,16–17,62) *p<0,05 class IV vs II; **p<0,05 Class IV vs II.Table 2. AGT gene expression in biopsies according proteinuria levels Proteinuria ≤0,5 Proteinuria >0,5 p ΔCt AGT Biopsies 3,60 (3,34–4,41) 4,79 (3,73–6,39) 0,04 Conclusions In the present study we found a potential utility of AGT mRNA levels in samples of active vs remission LN patients. Prospective studies are needed for confirming these results. References Yajuan Li et al. Biomarkers Profiling for Lupus Nephritis. Genomics Proteomics Bioinformatics 11; 158–165, 2013. Eriguchi M et al. Assessment of urinary angiotensinogen as a marker of podocyte injury in proteinuric nephropathies. Am J Physiol Renal Physiol 310: F322–F333, 2016. Mas V et al. Establishing the molecular pathways involved in chronic allograft nephropathy for testing new non-invasive diagnostic markers. Transplantation. 83:448–57, 2007. Disclosure of Interest None declared

AB0010 Angiogenesis Involved in Early Stages of Lupus Nephritis: Table 1.

Background Lupus Nephritis (LN) is one of the most frequent manifestations of SLE and can be present in 60% of SLE patients. It has been shown that structural changes and inflammatory infiltrate associated with LN contribute to a hypoxic state which induces angiogenesis [1]. Angiogenesis is a complex process that requires interaction between different cell types, the extracellular matrix, several cytokines and growth factors. Objectives We proposed to study gene expression levels of angiogenic factors such as VEGF-A (Vascular Endothelial Growth Factor), VCAM-1 (Vascular cell adhesion molecule 1), TGF-β (Transforming Growth Factor), SEL-E (Selectin-E), ANGPT1 (Angiopoietin 1), AGT (Angiotensinogen) and END (Endostatin) in kidney biopsies from LN patients and their relationship with LN severity. Methods Thirty two kidney biopsies samples were obtained from 32 LN patients and then classified according to ISN/RPS scoring system [3] in two groups, Class I/Class II biopsies (n=11, 10F/1M, age: 34.64±15.10, range: 21-72) and Class IV biopsies (n=21, 16F/5M, age: 30.95±11.36, range: 17-64). RNA was isolated using TRIzol-Chloroform technique and then was reverse-transcribed using random primers. Gene expression level of pro-angiogenic factors: VEGF, VCAM-1, TGF-β, SEL-E, ANGPT-1 and anti-angiogenic factors: AGT and END were evaluated using Quantitative Real Time PCR (QPCR). The threshold cycle (Ct) scores were averaged for calculations of relative expression values. The Ct scores were normalized against Ct scores by subtracting β2Microglobuline (β2M) control, or ΔCt=Ct,gene- Ct,B2M. To test for differential gene expression between groups, a two sample T-test was performed to compare the ΔCt in the two groups. Results ΔCt is inversely proportional to the gene expression level. Significant differences between groups were found in pro-angiogenic genes VEGF-A (p=0,05), VCAM-1 (p=0,05) and in anti-angiogenic gene END (p=0,05) (Table 1). There were not statistically significant differences in the expression of AGT, ANGPT-1, SEL-E and TGF-β between groups. After T test, we evaluated data from ΔCt analysis observing that the levels of mRNA of VEGF-A and VCAM-1 in Class I/II group were higher than those from Class IV group. Conversely, END gene expression was decreased in Class I/II group.Table 1. Gene expression levels of angiogenic factors between groups Gene Class I/II Class IV p value VEGF-A 1,68±1,72 3,13±2,02 0,05 VCAM-1 5,62±3,45 8,09±2,95 0,05 TGF-β 6,36±1,80 6,58±1,57 0,73 SEL-E 14,24±3,44 15,63±4,64 0,45 ANGPT-1 9,64±2,70 10,53±2,12 0,50 AGT 4,48±1,85 4,45±1,01 0,92 END 6,48±6,13 3,19±3,21 0,05 Conclusions In the present cross-sectional study, increased levels of VEGF-A and VCAM-1 and decreased levels of END were observed in biopsies classified as Class I and II. These findings could be associated with angiogenesis process in early stages of the disease. Angiogenesis in progression to LN deserves further evaluation. References Feliers D. Vascular Endothelial Growth Factor as a prognostic marker of Lupus Nephritis. Kidney Int; 75:1251-1253. 2009. Weening JJ. et al. The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int: 65 (2):521-30. 2004. Disclosure of Interest None declared

AB0008 Blys/April Gene Expression in Synovial Fluid and Blood in Rheumatoid Arthritis: Table 1.

Background The B Lymphocyte Stimulator (BLyS) and A PRoliferation-Inducing Ligand (APRIL) signaling pathway has an important role in the selection, maturation and survival of B cells. Dysregulation of BLyS/APRIL is involved in the pathogenesis of B-cell related autoimmune diseases including Rheumatoid Arthritis (RA). Synovial fluid cells are highly activated in patients with active RA inducing lymphocyte proliferation, expression of cell surface molecules, cytokine and auto antibody secretion. Objectives The purpose of this work is to evaluate Synovial Fluid (SF) and Peripheral Blood (PB) mRNA expression of BLyS and APRIL in RA patients. Methods SF and PB were obtained and classified in two groups, Group I: active RA patients with DAS 28 score >5.1 (n=11, 8F/3M, age: 56,2±20,9; range: 17-84) and Group II, control: Osteoarthritis (OA, n=20, 13F/7M, age: 69,8±9,5, range: 47-84). Levels of BLyS and APRIL expression were evaluated using Quantitative Real Time PCR (QPCR). All amplifications were carried out in duplicate and threshold cycle (Ct) scores were averaged for calculations of relative expression values. The Ct scores were normalized against Ct scores by subtracting the corresponding β2Microglobuline (β2M) control, or ΔCt=Ct,gene- Ct,B2M. To test for differential gene expression between groups, a two sample t-test was performed to compare the DCt in the two groups. Results BLyS and APRIL gene expression is shown in Table 1. Table 1. Levels of BLyS and APRIL gene expression in the two groups Group BLyS (Mean ΔCt) p value APRIL (Mean ΔCt) p value SF Group I 5,883 p=0,002 7,195 p=0,024 SF Group II 7,878 9,829 PB Group I 9,315 p=0,830 9,119 p=0,514 PB Group II 8,926 8,549 ΔCt is inversely proportional to the gene expression level. Analysis of PB showed no significant difference in gene expression between RA and OA. In SF, we observed a significant difference for BLyS and APRIL expression between Group I vs. Group II (p=0,002 and p=0,024 respectively). After t test, we evaluated data from

AB0018 Tubulointerstitial injury in lupus nephritis and gene expresion of KIM-1

Δ

THU0020 Angiogenesis in Rheumatoid Arthritis: VEGF Expression in Synovial Fluid

Ct analysis observing that in SF, mRNA of BLyS and APRIL in Group I was higher than those from Group II. Conclusions Gene expression of BLyS/APRIL in PB does not correlate with expression in SF. Increased BLyS and APRIL expression in active RA SF can be linked to B cell activation and maintenance in RA synovium. References Moura RA, et al. BAFF and TACI gene expression are increased in patients with untreated very early Rheumatoid Arthritis. The journal of Rheumatology 2013. La Cava A. Targeting the BLyS-APRIL signaling pathway in SLE. Clin Immunol 2013. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2971

THU0019 BLyS Expression in Synovial Fluids from Rheumatoid Arthritis Patients

Background The extent of tubular lesions and recruitment of inflammatory cells is belived to be and important predictor of renal function in immune- mediated glomerulonephritis (1) such as Lupus Nephritis (LN). The response of the renal tubules to proteinuria is implicated in progression of renal disease (2). Kidney Injury Molecule 1 (KIM-1), a recently discovered transmembrane tubular protein, is markedly induced in acute kidney injury and chronic kidney disease. KIM-1 is an ideal biomarker because is not expressed in normal kidney but specifically expressed in injured proximal tubular cells and such expression persist until the damage cells recovered (3). Objectives The role of KIM-1 in LN remains elusive. In this study, we examined the correlation between gene expression of KIM-1 in the urinary sediment and biopsy of patients (P) with NL diagnosis, and the relationship between KIM-1 expression and urine Protein/Creatinine ratio (P/C). Methods Twenty two kidney biopsies and 28 urine samples from 20 P with LN (17 F/3 M, age 33,55±12,28; Range: 15-72) were evaluated. Kidney biopsies were classified according to ISN/RPS scoring system (4). Urine samples from LN patients were divided as P/C <1 (Group I, N=12) and P/C >1 (Group II, N=16), and urine samples from healthy individuals (Group III, N=17) were analized as control. Levels of gene expression of KIM-1 were evaluated using Quantitative Real Time PCR (QPCR). All amplifications were carried out in duplicate and threshold cycle (Ct) scores were averaged for calculations of relative expression values. The Ct scores were normalized against Ct scores by subtracting the corresponding β2Microglobuline (β2M) control, or DCt=Ct,gene- Ct,B2M. A Spearman’s rank-order correlations (r) was used to test associations between gene expression levels in biopsy and urine pairs samples. To test for differential gene expression between groups a variance analysis (ANOVA) was performed. Results We observed a significant correlation between biopsy and urine, Spearman r=0,6838 (p=0.0005). There were a statistically significant difference in the expression of KIM-1 between groups (p=0,0110). After ANOVA test, we observed that the levels of mRNA of KIM-1 in Group II were higher than those from Group I and there were higher than Group III. Conclusions There was no expression of KIM-1 in normal urine. In LN, urinary KIM-1 gene expression is closely related to tissue KIM-1 and correlates with the severity of tubular interstitial injury. Quantitation of urinary KIM-1 is likely to be a nobel noninvasive and sensitive method for the evaluation of kidney injury in P with LN. References Zheng L, et al. Journal of Histochemistry and Cytochemistry. 56(5): 517-529, 2008. Hill G, et al. Kidney International. 60: 1893-1903, 2001. Hou W, et al. Transplantation Reviews. 24:143-146, 2010. Weening JJ, et al. Kidney International. 65 (2): 521-530, 2004. Disclosure of Interest None Declared

Evaluation of gene panel mRNAs in urine samples of kidney transplant recipients as a non-invasive tool of graft function

Background The expansion of synovial epithelium in Rheumatoid Arthritis (RA), and the subsequent pannus invasion of underlying cartilage and bone, needs an increase in the vascular supply to the synovium. Angiogenesis is recognised as a key event in the formation and maintenance of the pannus in RA. The pro-angiogenic cytokine, vascular endothelial growth factor (VEGF), has been demonstrated to have a central involvement in the angiogenic process in RA. Objectives We evaluated gene expression levels of a set of genes associated with angiogenesis in Synovial Fluid (SF) from RA patients compared with osteoarthritis patients. Methods Knee SF samples were classified in two groups, Group I: RA with DAS 28 score > 5.1, high disease activity (n=10, 7F/3M, age: 56,3 ± 20,9, range: 17-84) and Group II: Osteoarthritis (OA, n=18, 13F/5M, age: 70,5 ± 6,6, range: 58-86). Levels of gene expression of three genes previously associated with angiogenesis, including pro-angiogenic factors: VEGF and Angiopoietin 1 (ANGPT-1) and anti-angiogenic factor, Thrombospondin I (TSP1) were evaluated using Quantitative Real Time PCR (QPCR). All amplifications were carried out in duplicate and threshold cycle (Ct) scores were averaged for calculations of relative expression values. The Ct scores were normalized against Ct scores by subtracting the corresponding β2Microglobuline (β2M) control, or ΔCt=Ct,gene- Ct,B2M. To test for differential gene expression between groups, a two sample t-test was performed to compare the ΔCt in the two groups. Results Gene expression level is showed in table 1. Conclusions In the present cross-sectional study, increased levels of VEGF and decreased levels of TSP-1 were observed in RA patients. These gene expression results might be associated with increased angiogenesis in RA patients with high disease activity. References Paleolog EM. Angiogenesis in rheumatoid arthritis. Supplement Review. Arthritis Res 2002, 4 (3): S81-S90. Afuwape AO et al. The role of the angiogenic molecule VEGF in the pathogenesis of rheumatoid arthritis. Histol Histopathol 2002, 17: 961-972. Disclosure of Interest None Declared

Clinical Utility of a Modified qRT-PCR for Trypanosoma Cruzi Detection in Transplant Patients

Background BLyS is a novel TNF family ligand, mainly secreted by myeloid cellsandsynovial fibroblasts that exerts profound effects on B cells leading to their maturation and increased survival. Local expression of BLyS in inflamed joints from Rheumatoid Arthritis (RA) patients may contribute to the development and progression of disease. Objectives We evaluated local gene expression of BLyS in RA patients with disease activity. Methods Synovial fluids and Peripherical Blood (PB) were obtained from RA patients and were classified in two groups, Group I: RA evaluated by DAS 28 score as high disease activity (n=10, 7F/3M, age: 56,3 ± 20,9, range: 17-84) and Group II: Osteoarthritis (OA, n=18, 13F/5M, age: 70,5 ± 6,6, range: 58-86). Levels of BLyS expression were evaluated using Quantitative Real Time PCR (QPCR). All amplifications were carried out in duplicate and threshold cycle (Ct) scores were averaged for calculations of relative expression values. The Ct scores were normalized against Ct scores by subtracting the corresponding β2Microglobuline (β2M) control, or ΔCt=Ct,gene- Ct,B2M. To test for differential gene expression between groups, a two sample t-test was performed to compare the ΔCt in the two groups. Results BLyS gene expression is shown in Table 1. Conclusions Increased BLyS expression in active RA SF can be linked to B cell activation and proliferation in RA synovium. References Tan SM et al. Local Production of B lymphocyte Stimulator Protein and APRIL in Arthritic Joints of Patients with Inflammatory Arthritis. Arthritis and Rheumatism. 48 (4):982-992 (2003). Kreuzaler M et al. Soluble BAFF Levles inversely correlate with Peripherical B cell numbers and the expression of BAFF receptors. J Immunol. 188: 497-503 (2012). Disclosure of Interest None Declared