Background Lupus nephritis (LN) is one of the most severe forms of Systemic Lupus Erythematosus (SLE). Given that the kidney is the main site of inflammation in LN, biomarkers in urine may reflect this inflammation more closely than those in the blood. Many groups have studied messenger RNA (mRNA) expression of urinary biomarkers in patients with SLE, investigating which is likely to be most helpful for monitoring renal disease activity. Endostatin (END) is a natural proteolytic fragment of collagen XVIII and has been known to have modulatory function in angiogenesis and inflammation. In contrast to the general angiogenic factors, the expression profiles and activity of angiogenic inhibitors like END in LN are not well defined. Objectives We proposed to study gene expression level of END in urine from LN patients. Methods A total of 45 patients, 36 with renal involvement and 9 non-renal were included. Urine samples from active patients were divided according Protein Creatinin ratio (P/C) as: Group 1, P/C <1 (n=18) and Group II, P/C>1 (n=18), and non renal patients: Group III (n=9). Levels of gene expression of END were measured 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 by subtracting the corresponding β2Microglobuline (β2M) control, or ΔCt=Ct,gene − Ct,B2M. To test for differential gene expression between groups a variance analysis (ANOVA) and t test was performed. Results ΔCt is inversely proportional to the gene expression level. Urinary END was significantly decreased in active renal SLE patients compared with no-renal SLE patients (Table 1). Among active renal patients, END gene expression was elevated in Group I compared with Group II (test t, p=0.0192). Table 1. Levels of END gene expression between groups (ANOVA, p=0.0327) END (mean ΔCt) SLE patients p=0.0327 Active renal No-renal Group I (P/C <1) Group II (P/C >1) Group III 6,521 9,572 5,414 Conclusions Urinary END had the capacity to discriminate patients with active renal SLE from those with no-renal disease and exhibited the lowest urinary level in patients with P/C >1. This study provides evidence that measuring urinary END could be an important new biomarker in patients with SLE without renal activity. References Angiogenesis and hypoxia in the kidney. Tanaka T et al. Nat. Rev. Nephrol. 9, 211-222 (2013). Can measuring urinary biomarkers improve the management of Lupus Nephritis? Rahman A. Arthritis Research & Theraphy. 14, 127 (2012). Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5600

BackgroundLupus nephritis (LN) is one of the most frequent and serious complications in the patients with systemic lupus erythematosus. Several studies have identified risk factors for poor kidney prognosis in patients with SLE, including age, sex, hypertension, decreased estimated GFR (eGFR), proteinuria, and renal pathologic types.1 Biopsy allows classifying the type of renal involvement, assessing its activity, and thus guiding the therapeutic behaviours. It has been shown that structural changes and inflammatory infiltrate associated with LN contribute to a hypoxic state which induces angiogenesis.2 Herein, it was hypothesised that differential expression of angiogenic genes could classify Lupus Nephritis Patients (LNP) with the same histological score but different “clinical status”.ObjectivesTo investigate if there is a differential angiogenic gene expression in biopsies of LNP under the same histological classification but different “clinical status” measured by eGFR.MethodsTwenty four kidney biopsies samples classified according to ISN/RPS scoring system as Class IV from 24 LNP were divided into eGFR <60 ml/min (n=10, age: 31.00±10.93, range: 17–46) and eGFR >60 ml/min (n=14, age: 32.64±11.34, range: 21–64). RNA was isolated using TRIzol-Chloroform technique and then was reverse-transcribed using random primers. Gene expression level of pro-angiogenic factors: VCAM-1, VEGF, TGF-β and ANGPT-1 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 normalised by subtracting β2Microglobuline (β2M) control, or ΔCt=Ct, gene- Ct,β2M. To test for differential gene expression between groups, a two sample T-test was performed to compare the ΔCt in the two groups.ResultsΔCt is inversely proportional to the gene expression level. Significant differences between groups was found in VEGF-A gene (p=0.0326), where the greatest expression corresponding to eGFR <60 ml/min group. However, there were not statistically significant differences in VCAM-1, ANGPT1 and TGF-β expression (table 1). Particularly TGF-β, a proangiogenic and profibrotic gene showed a uniform expression level in both groups.Abstract AB0018 – Table 1Levels of gene expression and laboratory parameters in eGFR <60 ml/min and eGFR >60 mil/min expressed as ΔCt values.GeneeGFR<60 ml/mineGFR>60 ml/minp value n=10n=14 VCAM-15,021±17686,248±32640,3165VEGF-A3,414±11614,651±14090,0326TGF-β6,168±10116,699±13510,3421ANGPT110,330±22949,891±21130,5007ConclusionsIn the present cross-sectional study, increased levels of VEGF-A were observed in biopsies Class IV from LNP with eGFR <60 ml/min. These findings suggest a differential gene expression that may be associated with an impaired renal function, reflected by eGFR.References[1] Thong B, Olsen NJ. Systemic lupus erythematosus diagnosis and management. Rheumatology (Oxford)2017;56(suppl_1):i3–13.[2] Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature2011;19:298–307.[3]...

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 $Δ $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

BackgroundLupus nephritis (LN) is a severe complication of Systemic Lupus Erythematosus (SLE). Non-invasive biomarkers are needed for diagnosis of LN and to identify patients at risk of a renal flare (1). Thus the presence of biomarkers associated with inflammation, tissue damage or cell activation in the urine of patients with LN may be a useful tool in the evaluation of LN patients.The glomerular filtration rate (GFR) is considered the best overall index of renal function in health and disease. Because GFR is difficult to measure in clinical practice, most clinicians estimate the GFR (eGFR) from the serum creatinine concentration (2).B Lymphocyte Stimulator (BLyS) is a cytokine that fosters B cell activation, antibody production, B cell - T cell interaction and plasma cell survival. These events have been demonstrated to play a role in patients with LN (3).ObjectivesWe evaluated urinary levels of BLyS as biomarker for LN and their relationship with eGFR.MethodsUrine samples (n=86) were obtained from LN patients and classified in two groups: patients with eGFR >60 (GFRhigh, n=68, 62F/6M, age: 34.07±13.24) and patients with eGFR ≤60 (GFRlow, n=18, 14F/4M, age: 35.22±13.76). RNA from urine samples was isolated using TRIzol-Chloroform technique and then reverse-transcribed using random primers. 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 DCt=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.ResultsDCt is inversely proportional to BLyS's expression. We evaluated data from ΔCt analysis observing that mRNA levels of BLyS in eGFRlow (6.193±1.787) were higher than those from eGFRhigh (7.564±2.326), with a statistically significant difference between groups (p=0.0288).eGFRloweGFRhigh BLyS (ΔCt)6.193±1.7877.564±2.326p=0.0288ConclusionsIn the present cross-sectional study, increased levels of BLyS were observed in patients with eGFR ≤60. These gene expression results might be linked to B cell activation and proliferation in kidney and thus in urine samples. Combination of eGFR and BLyS appears to be a good biomarker.References Rahman A. Can measuring urinary biomarkers improve the management of lupus nephritis? Arthritis Research & Therapy 2012, 14:127.Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med. 1999 Mar 16; 130 (6):461–70.Phatak S, Chaurasia S, Mishra SK, Gupta R, Agrawal V, Aggarwal A, Misra R. Urinary B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL): potential biomarkers of active lupus nephritis. Clin Exp Immunol. 2016....

BackgroundLupus 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.ObjectivesTo investigate AGT expression in biopsies and urines from LN patients.Methods32 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.Results26 (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 samplesClass I/Class IIClass IVClass V/VIp Normal BiopsiesBiopsiesBiopsiesBiopsies n=3n=6n=12n=10p ΔCt AGT Biopsies*5,573,675,344,350,02(3,60–5,57)(2,19–5,37)(4,75–10,93)(3,45–4,57)ΔCt AGT Urines**14,1111,1916,7715,060,01(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 levelsProteinuria ≤0,5Proteinuria >0,5p ΔCt AGT Biopsies3,60 (3,34–4,41)4,79 (3,73–6,39)0,04ConclusionsIn 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 InterestNone declared