Urinary metabolomic profiling of a cohort of Colombian patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune and multisystem disease with a high public health impact. Lupus nephritis (LN), commonly known as renal involvement in SLE, is associated with a poorer prognosis and increased rates of morbidity and mortality in patients with SLE. Identifying new urinary biomarkers that can be used for LN prognosis or diagnosis is essential and is part of current active research. In this study, we applied an untargeted metabolomics approach involving liquid and gas chromatography coupled with mass spectrometry to urine samples collected from 17 individuals with SLE and no kidney damage, 23 individuals with LN, and 10 clinically healthy controls (HCs) to identify differential metabolic profiles for SLE and LN. The data analysis revealed a differentially abundant metabolite expression profile for each study group, and those metabolites may act as potential differential biomarkers of SLE and LN. The differential metabolic pathways found between the LN and SLE patients with no kidney involvement included primary bile acid biosynthesis, branched-chain amino acid synthesis and degradation, pantothenate and coenzyme A biosynthesis, lysine degradation, and tryptophan metabolism. Receiver operating characteristic curve analysis revealed that monopalmitin, glycolic acid, and glutamic acid allowed for the differentiation of individuals with SLE and no kidney involvement and individuals with LN considering high confidence levels. While the results offer promise, it is important to recognize the significant influence of medications and other external factors on metabolomics studies. This impact has the potential to obscure differences in metabolic profiles, presenting a considerable challenge in the identification of disease biomarkers. Therefore, experimental validation should be conducted with a larger sample size to explore the diagnostic potential of the metabolites found as well as to examine how treatment and disease activity influence the identified chemical compounds. This will be crucial for refining the accuracy and effectiveness of using urine metabolomics for diagnosing and monitoring lupus and lupus nephritis.

Comparison of Urinary Protein/Creatinine Ratio as an Alternative to 24-h Proteinuria in Lupus Nephritis: TUNARI Study.

BACKGROUND: Lupus nephritis (LN) occurs in approximately 50% of people with systemic lupus erythematosus (SLE). The 24-h proteinuria (gold standard) is measured among other tests for the control and monitoring of LN activity. This study investigates the use of the protein/creatinine ratio (PCR) as an alternative for the detection of proteinuria and its accuracy compared to the gold standard in a predominantly non-white population. METHODS: This was a prospective study conducted in Salvador, Brazil, between December 2021 and May 2022. We invited adult patients diagnosed with SLE and LN, regardless of their disease activity. The estimation of the PCR and 24-h proteinuria was performed using conventional methods. The analysis used was Spearman's r correlation coefficient (rs), coefficient of determination (r2), and concordance by the Bland-Altman method. A specific sensitivity was measured by the ROC curve with its respective cut-off by the Youden Index. RESULTS: We compared 112 samples of 75 patients with LN, with a mean age of 34.5 ± 11.8 years. Of these patients, 85% were women, 87.9% were non-white. A high degree of correlation was observed between PCR with 24-h proteinuria (rs = 0.77 and r2 = 0.59). The ROC analysis shows an area under the curve of 0.92 and the cut-off point calculated by the Youden Index was 0.78 with a sensitivity of 90.0% and specificity of 82%. However, the Bland-Altman graph indicated decreasing concordance as the degree of proteinuria increased, despite showing concordance at high levels of proteinuria. CONCLUSION: The PCR shows high sensitivity to follow-up patients with LN when compared with 24-h proteinuria. Our findings suggest that PCR is a useful parameter for the evaluating and monitoring patients in complete remission. However, in cases of partial remission, the utility of PCR is limited.

Comparison of urinary parameters, biomarkers, and outcome of childhood systemic lupus erythematosus early onset-lupus nephritis.

BACKGROUND: Urinary parameters, anti-dsDNA antibodies and complement tests were explored in patients with childhood-Systemic Lupus Erythematosus (cSLE) early-onset lupus nephritis (ELN) from a large multicenter cohort study. METHODS: Clinical and laboratory features of cSLE cases with kidney involvement at presentation, were reviewed. Disease activity parameters including SLEDAI-2 K scores and major organ involvement at onset and follow up, with accrued damage scored by SLICC-DI, during last follow up, were compared with those without kidney involvement. Autoantibodies, renal function and complement tests were determined by standard methods. Subjects were grouped by presence or absence of ELN. RESULTS: Out of the 846 subjects enrolled, mean age 11.6 (SD 3.6) years; 427 (50.5%) had ELN. There was no significant difference in the ELN proportion, according to onset age, but ELN frequency was significantly higher in non-Caucasians (p = 0.03). Hematuria, pyuria, urine casts, 24-h proteinuria and arterial hypertension at baseline, all had significant association with ELN outcome (p < 0.001). With a similar follow up time, there were significantly higher SLICC-DI damage scores during last follow up visit (p = 0.004) and also higher death rates (p < 0.0001) in those with ELN. Low C3 (chi-square test, p = 0.01), but not C3 levels associated significantly with ELN. High anti-dsDNA antibody levels were associated with ELN (p < 0.0001), but anti-Sm, anti-RNP, anti-Ro, anti-La antibodies were not associated. Low C4, C4 levels, low CH50 and CH50 values had no significant association. High erythrocyte sedimentation rate (ESR) was associated with the absence of ELN (p = 0.02). CONCLUSION: The frequency of ELN was 50%, resulting in higher morbidity and mortality compared to those without ELN. The urinary parameters, positive anti-dsDNA and low C3 are reliable for discriminating ELN.

Utility of urinary transferrin and ceruloplasmin in patients with systemic lupus erythematosus for differentiating patients with lupus nephritis. / Transferrina y ceruloplasmina en orina de pacientes con lupus eritematoso sistémico. ¿Son útiles para diferenciar pacientes con nefritis lúpica?

BACKGROUND AND OBJECTIVE: Diagnosis of lupus nephritis (LN) is usually based on renal biopsy, which is an invasive technique that involves multiple risks. Therefore, different biomarkers have emerged as alternatives for the diagnosis of LN. Nonetheless, studies regarding urinary biomarkers in Latin American patients are limited. The objective of this study was to assess the diagnostic value of urinary transferrin and ceruloplasmin to differentiate patients who have renal involvement from those who do not. MATERIALS AND METHODS: Systemic lupus erythematosus (SLE) patients that met the revised American College of Rheumatology (ACR) classification criteria were recruited. Patients with another autoimmune disease, active infection (urinary tract or systemic infection), renal replacement therapy, human immunodeficiency virus infection or pregnancy were excluded. A urine sample was collected from each patient. LN was diagnosed according to ACR criteria. The activity and chronicity of LN were measured using the Austin indices. Urinary transferrin and ceruloplasmin levels were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits. Mann-Whitney U test and Student's t-test were used to compare data. Spearman's rank correlation was used to determine associations. Lastly, receiver operating characteristic (ROC) curves were created. RESULTS: The study involved 120 SLE patients. In all, 85% were female, 76% mestizo, the mean age was 32.8±12.1years and mean systemic lupus erythematosus disease activity index (SLEDAI) was 8.4±8.9; 64% had renal involvement. Urinary levels of the two biomarkers were significantly higher in patients with LN compared to those without LN. Similarly, urinary levels of both biomarkers were significantly higher in patients with active LN compared to those with inactive LN. Furthermore, urinary transferrin levels were significantly higher in Afro-Latin American patients. On the other hand, urinary transferrin levels correlated with SLEDAI and proteinuria, and transferrin and ceruloplasmin levels correlated with each other. The diagnostic value of ROC curves for these urinary biomarkers for LN were good. CONCLUSIONS: In our cohort of SLE patients, we found that transferrin and ceruloplasmin were potential biomarkers for LN, and can even differentiate active LN.

Hemoglobinuria paroxística nocturna. el rol de la anhidrasa carbónica-i urinaria en la hemólisis intravascular / Peroxysmal nocturnal hemoglobinuria: the role of urinary carbonic anhydrase-I in intravascular haemolysis

La Hemoglobinuria Paroxística Nocturna (HPN) se caracteriza por hemólisis intravascular crónica mediada por complemento. Cuando se produce la hemolisis se libera a circulación Anhidrasa Carbónica- I (AC-I), una enzima que se halla en alta concentración en el eritrocito y por su bajo peso molecular filtra por el glomérulo. El objetivo del presente trabajo fue detectar la excreción de la AC-I en orina de pacientes con HPN por Electroforesis Bidimensional de Utilidad Clínica (2D UC), y compararla con otras causas de hemólisis, de origen renal y postrenal. Se evaluaron 8 pacientes con HPN sin tratamiento con eculizumab un inhibidor del C5 del complemento, y 5 de ellos postratamiento, 12 orinas de pacientes con nefritis lúpica y 10 orinas de pacientes con hemólisis postrenal. La AC-I puede estar presente en la orina, en los tres grupos, sin embargo la relación AC-I/Hemoglobina en la hemólisis intravascular está invertida en comparación con la hemolisis glomerular y post-renal. Los pacientes con HPN tratados con eculizumab no presentan AC-I, y sería de utilidad en el seguimiento de los pacientes tratados con el inhibidor del C5, para evidenciar posibles escapes hemolíticos.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is characterized by chronic complement mediated haemolysis. In these conditions it might be expected that carbonic anhydrase-I (AC-I) would be liberated into the plasma and excreted in the urine, by its high concentration in the erythrocyte and low molecular weight. The objective of the present study was to detect the urinary excretion of AC-I from patients with PNH by wodimensional clinical utility electrophoresis (2D UC) and to compare it with other causes of renal and post-renal haemolysis. We evaluated 8 patients with PNH without eculizumab, a complement C5 inhibitor, 5 of them posttreatment, 12 urine of patients with lupus nephritis and 10 urine of patients with post-renal hemolysis. AC-I may be present in the urine, in all three groups, however, the AC-I/Haemoglobin ratio in intravascular haemolysis is reversed compared to glomerular and post-renal haemolysis. Patients with PNH treated with eculizumab do not have AC-I and would be useful in monitoring patients treated with the C5 inhibitor to evidence possible haemolytic leaks.

Laminin-1 (LM-111) in preeclampsia and systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of antibodies. SLE has been associated with placental pathology, a finding that is also the determinant in preeclampsia (PE). Genetic evidence and serologic reports suggest laminin-1 (LM-111) as an immunogenic molecule and its polymorphic gene as a candidate gene for both disorders. OBJECTIVE: To evaluate the association between LAMA1 (rs543355) and LAMC1 (rs20563) polymorphisms and the presence of SLE and PE as well as to determine serum levels of anti-LM-111 autoantibodies in the PE group. METHODS: Group A: 169 women with PE and 172 healthy pregnant women. Group B: 204 women with SLE and 204 healthy women. Anti-LM-111 for group A was measured by ELISA and the genotyping was done by using a PCR system. RESULTS: Group A: Levels of anti-LM-111 was similar in women with PE and the control group (p = 0.3). The allelic frequencies and genotypes did not show statistically significant differences for LAMA1 and LAMC1 polymorphisms. Group B: Significant differences between SLE patients and controls for rs543355 polymorphism were not observed. Nevertheless, LAMC1 rs20563 A-allele provided protection against the development of SLE (OR 0.73, 95%CI 0.55-0.96). CONCLUSIONS: Serum levels of anti-LM-111 at the third trimester of gestation do not seem to have any direct relationship with the presence of PE, and the SNPs evaluated are not associated with the risk of developing this disorder. LAMC1 polymorphism could be a protective factor for SLE.

Renal function assessment in patients with systemic lupus erythematosus.

Few studies have evaluated the glomerular filtration rate (GFR) in patients with systemic lupus erythematosus (SLE). Even though the National Kidney Foundation (NKF) suggests using the equations to estimate GFR, rheumatologists continue using creatinine clearance (CCl). The main objective of our study was the assessment of different equations to estimate GFR in patients with SLE: Simplified MDRD study equation (sMDRD), CCl, Cockcroft Gault (CG), CG calculated with ideal weight (CGi), Mayo Clinic Quadratic (MCQ), and Chronic Kidney Disease Epidemiology Collaboration Equation (CKD-EPI). CKD-EPI was considered as the reference standard, and it was compared with the other equations to evaluate bias, correlation (r), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), percentage of measurement of GFR between 70-130% of GFR measured through CKD-EPI (P30) and to compute the ROC curves. Adequacy of the 24-h urine collection was evaluated. To classify patients into GFR < 60 ml/min/1.73 m(2), the best sensitivity and NVP were obtained with sMDRD: the best PPV and specificity with MCQ. P30 was 99.3% with sMDRD, 77.5% CCl, 91.7% CG, 96.7% CGi, and 77.2% with MCQ. The lowest bias was for sMDRD and the highest for CCl. Only 159 (52.6%) urine collections were considered adequate, and when these patients were re-evaluated, the statistical results improved for CCl. CGi was better in general than CG. CCl should not be considered as an adequate GFR estimation. Ideal weight is better than real weight to calculate GFR through CG in patients with SLE.

Disease activity, proteinuria, and vitamin D status in children with systemic lupus erythematosus and juvenile dermatomyositis.

OBJECTIVE: To evaluate relationships among vitamin D, proteinuria, and disease activity in pediatric systemic lupus erythematosus (SLE) and juvenile dermatomyositis (JDM). STUDY DESIGN: Multiple linear regression was used to associate subject-reported race, sunscreen use, and vitamin D intake with physician-assessed disease activity and serum 25-hydroxyvitamin D (25[OH]D) in 58 subjects with pediatric SLE (n=37) or JDM (n=21). Serum 25(OH)D was correlated with urinary vitamin D binding protein/creatinine ratio (DBP/C) and other indicators of proteinuria. RESULTS: Serum 25(OH)D levels in subjects with SLE were inversely associated with the natural log of urinary DBP/C (r=-0.63, P<.001) and urine protein to creatinine ratio (r=-0.60, P<.001), with an adjusted mean 10.9-ng/mL (95% CI, 5.1-16.8) decrease in 25(OH)D for those with proteinuria. Excluding subjects with proteinuria, serum 25(OH)D levels were inversely associated with disease activity in JDM, but not in SLE. Overall, 66% of all subjects were taking concurrent corticosteroids, but this was not associated with 25(OH)D levels. CONCLUSIONS: Low serum 25(OH)D in patients with SLE is associated with proteinuria and urinary DBP. Vitamin D deficiency is associated with disease activity in patients with JDM and SLE; this relationship in SLE may be confounded by proteinuria.

Are urinary levels of free light chains of immunoglobulins useful markers for differentiating between systemic lupus erythematosus and infection?

INTRODUCTION: Active systemic lupus erythematosus (SLE) and infection are often hard to distinguish. We evaluated the urinary levels of free light chains of immunoglobulins (urFLCIg) as a possible laboratory marker to differentiate those conditions. METHODS: We evaluated 43 patients with lupus nephritis (16-63 years old), with or without concurrent infection (12 with infection), 14 with infectious disease without SLE and 20 with idiopathic Fanconi's syndrome. The Systemic Lupus Erythematosus Disease Activity Index was utilized to establish activity of disease. Levels of urFLCIg kappa and lambda were determined by an immunoenzymometric assay developed in our institution. In order to evaluate proximal tubular dysfunction which could be responsible for increased levels of urFLCIg, we determined the low-molecular-weight protein urRBP. RESULTS: Levels of urFLCIg in healthy volunteers (median kappa 1.57 mg/l; lambda 0.96 mg/l), inactive SLE (5.36; 4.93) and active SLE (11.82; 23.59) were significantly different; urFLCIg levels or urFLCIg/urRBP ratios could not separate patients with infection from those with SLE. CONCLUSION: Our data show that convoluted proximal tubular dysfunction was not responsible for the increase in urFLCIg levels. UrFLCIg determination was useful in the detection of SLE activity, but was unable to distinguish activity from infection in this condition.

Urinary neopterin quantification by reverse-phase high-performance liquid chromatography with ultraviolet detection.

Neopterin plays an important role in the malignant disease diagnostics. However, the methods employed in neopterin determination are generally difficult and/or time consuming. The aim of this work was to standardize a practical method to quantify neopterin using high-performance liquid chromatography-ultraviolet (HPLC-UV) and quantify it in patients with systemic lupus erythematosus (SLE). Urine was collected from healthy subjects (n= 49), patients with inactive (n= 15), active (n= 28), and highly active SLE (n= 6). The HPLC was performed using two coupled reverse-phase columns eluted with 150 mM sodium phosphate, pH 4.0, under a flow rate of 0.8 ml/min, with UV detector set at 353 nm and 100-fold diluted urines. The inter- and intra-assay studies presented an imprecision of 12.5% and 12.9% for quality controls of 3.94 and 1.1 micromol/ml, respectively. Recovery from 79.5% to 82% was observed throughout the assay's linear range. Subjects with active (874.2 +/- 165.38 micromol/mol creatinin) and highly active SLE (1753.8 +/- 453.9 micromol/mol creatinin) showed three- and sixfold increased neopterin levels, respectively, compared to subjects with inactive SLE (314.3 +/- 121.3 micromol/mol creatinin) and healthy subjects (294.6 +/- 178.6 micromol/mol creatinin) (P< 0.05). Briefly, the proposed method was precise, specific, and reproducible, not invasive and allows the urinary neopterin quantification only with UV detection.

Frequency of Mycoplasma hominis and Ureaplasma urealyticum infections in women with systemic lupus erythematosus.

Ureaplasma urealyticum (UU) and Mycoplasma hominis (MH) have been detected in the urine of women with systemic lupus erythematosus (SLE). We evaluated the presence of these mycoplasma in the endocervix of women presenting SLE. A total of 40 SLE patients (mean age 40.2 years), and 51 healthy women (mean age 30.9 years), were studied. Endocervical swabs were cultured in specific liquid media for MH or UU, detected by a quantitative color assay, and considered positive at >10(3) dilutions. Statistical analysis was performed using the two-tailed Fisher test. UU was detected in 52.5 % of patients and in 11.8% of controls (p= 0.000059). MH was detected in 20% of patients and 2% controls (p=0.003905). Both mycoplasmas were detected in 7.3% patients and 0% controls (p<0.000001). The results reported here corroborate the association of the mycoplasma infection and SLE. Thus, these agents may stimulate the production of autoreactive clones.

Frequency of Mycoplasma hominis and Ureaplasma urealyticum infections in women with systemic lupus erythematosus

Ureaplasma urealyticum (UU) and Mycoplasma hominis (MH) have been detected in the urine of women with systemic lupus erythematosus (SLE). We evaluated the presence of these mycoplasma in the endocervix of women presenting SLE. A total of 40 SLE patients (mean age 40.2 years), and 51 healthy women (mean age 30.9 years), were studied. Endocervical swabs were cultured in specific liquid media for MH or UU, detected by a quantitative color assay, and considered positive at >10(3) dilutions. Statistical analysis was performed using the two-tailed Fisher test. UU was detected in 52.5 % of patients and in 11.8% of controls (p= 0.000059). MH was detected in 20% of patients and 2% controls (p=0.003905). Both mycoplasmas were detected in 7.3% patients and 0% controls (p<0.000001). The results reported here corroborate the association of the mycoplasma infection and SLE. Thus, these agents may stimulate the production of autoreactive clones.

Ureaplasma urealyticum (UU) e Mycoplasma hominis (MH) têm sido detectados em urina de mulheres com lupus eritematoso sistêmico (LES). Avaliamos a presença destes mycoplasmas no endocervix de mulheres apresentando LES. Um total de 40 pacientes com LES (idade média de 40,2 anos), e 51 mulheres sadias (idade média de 30.9 anos), foram estudadas. Swabs do endocervix foram cultivados em meio líquido específico para MH e UU, detectados por teste colorimétrico quantitativo, considerando positivo diluições > 103 . Análise estatística foi feita usando teste de Fisher. UU foi detectado em 52,5% das pacientes e em 11,8% dos controles (p= 0.000059). MH foi detectado em 20% das pacientes e 2% dos controles (p=0.003905). Ambos mycoplasmas foram detectados em 7,3 % das pacientes e 0% dos controles (p<0.000001). Os resultados aqui reportados corroboram com a associação de infecção por mycoplasma e LES. Estes agentes podem estimular a produção de clones autoreativos.

Caracterização imunofenotípica dos leucócitos presentes na urina de pacientes com nefropatia lúpica em atividade / Immunophenotypic characterization of leukocytes present in urine of patients with active lupus nephropathy

O objetivo deste estudo foi determinar a expressão fenotípica dos leucócitos presentes na urina de pacientes com lúpus eritematoso sistêmico (LES) e nefrite em atividade. Foram selecionados 12 pacientes com glomerulonefrite membranosa. O critério utilizado para classificação de atividade sistêmica foi o SLEDAI (índice de atividade de doença para o LES), cuja média foi de 18,2 pontos. A presença de nefrite ativa foi definida por critérios histológicos, alterações do sedimento urinário, desenvolvimento recente de proteinúria e/ou perda aguda da função renal. A fenotipagem com anticorpos monoclonais (ACMo) e fosfatase alcalina, aplicada em lâminas com citocentrifugado de amostra da urina, foi o método empregado para a classificação das subpopulações de leucócitos urinários. Encontrou-se predomínio de células CD14 positivas (monócitos/macrófagos) nos pacientes com LES 56,8 por cento dos leucócitos urinários). A segunda subpopulação mais frequente foi a de células CD3 CD14 CD20-CD45 + (a maioria provavelmente granulócitos) e os linfócitos T representaram 8,1 por cento do total de leucócitos (teste de Wilcoxon com correção de Bonferroni - p < 0,016). Evidenciou-se uma associação positiva entre exsudação leucocitária glomerular e excreção urinária de linfócitos CD8 + (p = 0,01). Esses resultados reforçam os indícios sugestivos da participação das células de linhagem macrofágica na fisiopatologia da lesão renal proliferativa no LES

Decrease in serum antiphospholipid antibody levels upon development of nephrotic syndrome in patients with systemic lupus erythematosus: relationship to urinary loss of IgG and other factors.

PURPOSE: Having observed a decrease in antiphospholipid antibodies (aPL) upon the development of nephrotic syndrome, as well as a negative association between nephrotic syndrome and secondary antiphospholipid syndrome, in patients with systemic lupus erythematosus (SLE), we sought to determine if this could be due to urinary loss of aPL and/or other factors. SUBJECTS AND METHODS: IgG and IgM aPL as well as other autoantibodies were studied by enzyme-linked immunosorbent assay with cardiolipin as antigen in serum and urine from six patients with SLE who had elevated serum aPL levels and developed nephrotic syndrome (cases). For controls, we studied: (1) three SLE patients with nephrotic syndrome but low aPL levels; (2) three patients with non-SLE nephrotic syndrome; (3) three SLE patients with high-titer aPL but no proteinuria; and (4) 10 healthy volunteers. RESULTS: We found urinary IgG, but no IgM, aPL in all cases and in one control from Group 2. Serum IgG aPL had gradually decreased after the development of nephrotic syndrome and had become normal. IgM aPL had also decreased in the four patients who had elevated levels, having reached normal levels at the time of the study in two. There was an apparent correlation between serum and urine IgG aPL levels but not between urinary IgG aPL and total proteinuria. By Farr's method, we found no urinary anti-DNA despite high serum titers in three cases. The two cases and one of the controls in Group 1 who had serum antibodies to extractable antigens also had these antibodies in the urine. CONCLUSION: Urinary loss of IgG aPL during nephrotic syndrome does not completely explain the reduction in serum aPL, since IgM also decreases. There could also be decreased synthesis and/or increased catabolism of immunoglobulins.

El sedimento urinario en la nefropatía lúpica / Urinary sediment in lupus nephropathy

Se estudiaron 24 pacientes que cumplian los criterios preliminares de la Asociación Americana de Reumatología para LES y cuyas biopsias mostraron alteraciones compatibles con nefropatía lúpica. Todos ellos tenían al menos un sedimento urinario visto por un nefrólogo, estudio de la función renal valorada por depuración de creatinina, urea y creatinina sérica y proteinuria de 24 hora. Se concluye que el sedimento urinario realizado por el personal entrenado, puede ser de utilidad para predecir el grado de alteración histológica presente en la nefropatía lúpica