Non-invasive biomarkers of disease activity and organ damage in ANCA-associated vasculitis: a systematic review.

BACKGROUND: In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), histopathological assessment of affected tissue is often necessary for diagnosis and assessment of disease extent. There is a requirement for validated non-invasive biomarkers to avoid the need for serial tissue biopsies. METHODS: A systematic review of scientific databases from 2012 until present was performed to identify studies fulfilling the inclusion criteria. Studies were assessed for quality using the Strengthening the Reporting of Observational Studies in Epidemiology checklist for cohort, case-control and cross-sectional studies and the Risk of Bias Assessment tool for Non-randomised Studies, or the Cochrane Risk of Bias tool 2.0 for randomised controlled trials. A descriptive synthesis of the data for non-invasive (blood-based or urinary) biomarkers of AAV-related disease activity and organ damage was performed. RESULTS: Twenty-two high quality studies were included. These articles reported the value of blood-based and urinary biomarkers including anti-neutrophil cytoplasmic antibodies, immune cells, complement factors, gene expression profiles, cytokines, chemokines and other proteins in the assessment of disease activity and/or organ damage in patients with AAV. Many of these biomarkers involve the alternative complement pathway, neutrophil activation and macrophage activation. CONCLUSION: This is the first contemporary systematic review synthesising the value of non-invasive biomarkers of AAV-related disease activity and organ damage. The incorporation of individual markers in combined biomarker profiles might enhance clinical decision-making. Many unmet needs were identified; few studies involve oeosinophilic granulomatosis with polyangiitis and patients with childhood-onset AAV. Further validation of the candidate biomarkers is warranted in large prospective studies to bridge the existing knowledge gaps and apply precision health to systemic vasculitis.

Automated Computational Detection of Disease Activity in ANCA-Associated Glomerulonephritis Using Raman Spectroscopy: A Pilot Study.

Biospectroscopy offers the ability to simultaneously identify key biochemical changes in tissue associated with a given pathological state to facilitate biomarker extraction and automated detection of key lesions. Herein, we evaluated the application of machine learning in conjunction with Raman spectroscopy as an innovative low-cost technique for the automated computational detection of disease activity in anti-neutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis (AAGN). Consecutive patients with active AAGN and those in disease remission were recruited from a single UK centre. In those with active disease, renal biopsy samples were collected together with a paired urine sample. Urine samples were collected immediately prior to biopsy. Amongst those in remission at the time of recruitment, archived renal tissue samples representative of biopsies taken during an active disease period were obtained. In total, twenty-eight tissue samples were included in the analysis. Following supervised classification according to recorded histological data, spectral data from unstained tissue samples were able to discriminate disease activity with a high degree of accuracy on blind predictive modelling: F-score 95% for >25% interstitial fibrosis and tubular atrophy (sensitivity 100%, specificity 90%, area under ROC 0.98), 100% for necrotising glomerular lesions (sensitivity 100%, specificity 100%, area under ROC 1) and 100% for interstitial infiltrate (sensitivity 100%, specificity 100%, area under ROC 0.97). Corresponding spectrochemical changes in paired urine samples were limited. Future larger study is required, inclusive of assigned variables according to novel non-invasive biomarkers as well as the application of forward feature extraction algorithms to predict clinical outcomes based on spectral features.

The Clinical Application of Urine Soluble CD163 in ANCA-Associated Vasculitis.

BACKGROUND: Up to 70% of patients with ANCA-associated vasculitis (AAV) develop GN, with 26% progressing to ESKD. Diagnostic-grade and noninvasive tools to detect active renal inflammation are needed. Urinary soluble CD163 (usCD163) is a promising biomarker of active renal vasculitis, but a diagnostic-grade assay, assessment of its utility in prospective diagnosis of renal vasculitis flares, and evaluation of its utility in proteinuric states are needed. METHODS: We assessed a diagnostic-grade usCD163 assay in (1) a real-world cohort of 405 patients with AAV and 121 healthy and 488 non-AAV disease controls; (2) a prospective multicenter study of 84 patients with potential renal vasculitis flare; (3) a longitudinal multicenter cohort of 65 patients with podocytopathy; and (4) a cohort of 29 patients with AAV (with or without proteinuria) and ten controls. RESULTS: We established a diagnostic reference range, with a cutoff of 250 ng/mmol for active renal vasculitis (area under the curve [AUC], 0.978). Using this cutoff, usCD163 was elevated in renal vasculitis flare (AUC, 0.95) but remained low in flare mimics, such as nonvasculitic AKI. usCD163's specificity declined in patients with AAV who had nephrotic-range proteinuria and in those with primary podocytopathy, with 62% of patients with nephrotic syndrome displaying a "positive" usCD163. In patients with AAV and significant proteinuria, usCD163 normalization to total urine protein rather than creatinine provided the greatest clinical utility for diagnosing active renal vasculitis. CONCLUSIONS: usCD163 is elevated in renal vasculitis flare and remains low in flare mimics. Nonspecific protein leakage in nephrotic syndrome elevates usCD163 in the absence of glomerular macrophage infiltration, resulting in false-positive results; this can be corrected with urine protein normalization.

Assessment of the correlation of commonly used laboratory tests with clinical activity, renal involvement and treatment of systemic small-vessel vasculitis with the presence of ANCA antibodies.

BACKGROUND: The aim of the study was to assess the correlation of commonly used laboratory tests with clinical activity, degree of kidney involvement and treatment of systemic small-vessel vasculitis with the presence of ANCA antibodies. METHODS: The study included 28 patients with active AAV (BVAS ≥ 3). The following tests were performed: MPO-ANCA, PR3-ANCA, peripheral blood count, ESR, CRP, procalcitonin, creatinine, GFR, urea, albumin, fibrinogen, d-dimer, components of the C3 and C4 complement systems, urinalysis with sediment evaluation and diurnal proteinuria. The assessments were conducted twice: at study entry (A0) and after 6 months (A6) (BVAS = 0). RESULTS: At the time of inclusion in the study, the mean creatinine concentration was 3.39 mg/dl (GFR 33.17 ml/min/1.73 m²), after achieving remission in 11 patients (39.3 %) GFR remained below 30 ml/min/1.73 m², 4 patients (14.3 %) continued renal replacement therapy, and 3 patients (10.7 %) with advanced renal failure died. Microscopic hematuria occurred in 80.9 % of the studied population, withdrew in most patients, strongly correlated with renal involvement p < 0.001 and was not related to disease severity p = 0.147. CRP, ESR, fibrinogen, d-dimer, albumin and hemoglobin in the peripheral blood showed a strong correlation with the clinical activity of AAV and well identified severe patients. High procalcitonin concentrations correlated with a severe form of the disease, pulmonary involvement with respiratory failure and alveolar hemorrhage (mean 3.41 ng/ml, median 0.91 ng/ml, SD 7.62, p = 0.000), and were associated with the occurrence of infectious complications and the need to administer antibiotic therapy. ANCA antibodies were useful in the evaluation of patients with AAV, the amount of antibodies did not correlate with the severity of vasculitis (p = 0.685) and the results in many patients did not match the expected assumptions. CONCLUSIONS: CRP, ESR, fibrinogen, d-dimers, albumin and hemoglobin in the peripheral blood correlate well with the activity of vasculitis and identify severe patients. The resolution of microscopic hematuria suggests remission of the disease in the renal area. Procalcitonin may be slightly increased in patients with active AAV without infection, high concentrations are strongly associated with infectious complications. ANCA antibodies should always be interpreted in the context of the observed clinical symptoms.

Distinguishing active from quiescent disease in ANCA-associated vasculitis using attenuated total reflection Fourier-transform infrared spectroscopy.

The current lack of a reliable biomarker of disease activity in anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitis poses a significant clinical unmet need when determining relapsing or persisting disease. In this study, we demonstrate for the first time that attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy offers a novel and functional candidate biomarker, distinguishing active from quiescent disease with a high degree of accuracy. Paired blood and urine samples were collected within a single UK centre from patients with active disease, disease remission, disease controls and healthy controls. Three key biofluids were evaluated; plasma, serum and urine, with subsequent chemometric analysis and blind predictive model validation. Spectrochemical interrogation proved plasma to be the most conducive biofluid, with excellent separation between the two categories on PC2 direction (AUC 0.901) and 100% sensitivity (F-score 92.3%) for disease remission and 85.7% specificity (F-score 92.3%) for active disease on blind predictive modelling. This was independent of organ system involvement and current ANCA status, with similar findings observed on comparative analysis following successful remission-induction therapy (AUC > 0.9, 100% sensitivity for disease remission, F-score 75%). This promising technique is clinically translatable and warrants future larger study with longitudinal data, potentially aiding earlier intervention and individualisation of treatment.

Venous thromboembolism in ANCA-associated vasculitis: a population-based cohort study.

OBJECTIVE: To determine incidence rate and predictors of venous thromboembolic events (VTE) in a population-based cohort with ANCA-associated vasculitis (AAV). METHODS: The study comprised 325 patients diagnosed with AAV from 1997 to 2016. All cases of VTE from prior to vasculitis diagnosis to the end of the study period were identified. The BVAS was used to assess disease activity at diagnosis. Venous thromboembolisms occurring in a period beginning 3 months prior to AAV diagnosis were considered to be AAV-related. The standardized incidence ratio (SIR) and 95% CI of VTE were calculated using the incidence rate in the general population. RESULTS: Fifty-nine patients (18%) suffered 64 VTE events. Of these, 48 (81%) suffered AAV-related VTE [deep vein thrombosis (n = 23), pulmonary embolism (n = 18) and other (n = 9)]. The incidence rate of AAV-related VTE was 2.4 per 100 person-years (95% CI 1.7, 3.0) during 2039 person-years of follow-up. The incidence during the first 3 months post-AAV diagnosis was 20.4 per 100 person-years (95% CI 11.5, 29.4), decreasing to 8.9 (95% CI 0.2, 17.6) and 1.5 (95% CI 0.0, 3.5) in months 4-6 and months 7-12 post-AAV diagnosis, respectively. The SIR was 34.2 (95% CI 20.2, 48.1) for deep vein thrombosis and 10.4 (95% CI 5.6, 15.1) for pulmonary embolism. In multivariate Cox regression analyses, only age and BVAS were predictive of VTE. CONCLUSIONS: The incidence rate and SIR of AAV-related VTE is high, and higher early in the course of the disease. Vasculitis activity and age are positively associated with VTE.

Urinary Soluble CD163 and Disease Activity in Biopsy-Proven ANCA-Associated Glomerulonephritis.

BACKGROUND AND OBJECTIVES: ANCA-associated GN is a common cause of rapidly progressive GN, with high relapse rates. The early recognition of an ANCA-associated GN relapse is of importance to prevent loss of kidney function. Urinary soluble CD163 has been identified as a promising marker of active ANCA-associated GN. Previous studies, however, are limited by the lack of histologic data. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We analyzed urinary soluble CD163 in 95 patients with ANCA-associated vasculitis who underwent a kidney biopsy. In total, 125 kidney tissue sections (first kidney biopsy, n=67; repeated biopsy, n=58) with concurrent 24-hour urine samples were studied. Correlation analyses comparing urinary soluble CD163 levels and morphologic features of ANCA-associated GN were performed using Spearman rank correlation analysis. The diagnostic performance of biomarkers to detect relapsing ANCA-associated GN was evaluated using receiver operating characteristics curve analysis. RESULTS: High levels of urinary soluble CD163 were found in 96 (87%) of 110 biopsies with active ANCA-associated GN compared with one (7%) of 15 biopsies without active ANCA-associated GN and one (6%) of 17 healthy controls. Urinary soluble CD163 correlated with fibrinoid necrosis (Rho=0.48, P<0.001) and cellular crescents (Rho=0.70, P<0.001) on kidney biopsy. In repeated biopsies, urinary soluble CD163's sensitivity of 0.94 and specificity of 0.91 for the recognition of relapsing ANCA-associated GN appeared better than routine clinical measures. The presence of CD163+ cells in affected glomeruli confirmed urinary soluble CD163's origin. CONCLUSIONS: Urinary soluble CD163 is associated with active ANCA-associated GN and correlates with histologic features as seen in ANCA-associated GN. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2020_11_17_CJN07210520_final.mp3.

Sphingosine-1-phosphate receptor modulator FTY720 attenuates experimental myeloperoxidase-ANCA vasculitis in a T cell-dependent manner.

Sphingosine-1-phosphate (S1P) is a pleiotropic lysosphingolipid derived from the metabolism of plasma membrane lipids. The interaction between S1P and its ubiquitously expressed G-protein-coupled receptors (S1PR1-5) is crucial in many pathophysiological processes. Emerging evidence suggested a potential role for S1P receptors in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In the present study, we investigated the effects of three different S1P receptors modulators (FTY720, SEW2871 and TY52156) in a recognized rat model of experimental autoimmune vasculitis (EAV). The effects of treatments were evaluated with clinico-pathological parameters including hematuria, proteinuria, crescent formation, pulmonary hemorrhage, etc. In vitro functional studies were performed in a Jurkat T-cell line following stimulations of serum from myeloperoxidase-AAV patients. We found that only the FTY720 treatment significantly alleviated hematuria and proteinuria, and diminished glomerular crescent formation, renal tubulointerstitial lesions and pulmonary hemorrhage in EAV. The attenuation was accompanied by less renal T-cell infiltration, up-regulated mRNA of S1PR1 and down-regulated IL-1ß in kidneys, but not altered circulating ANCA levels, suggesting that the therapeutic effects of FTY720 were B-cell independent. Further in vitro studies demonstrated that FTY720 incubation could significantly inhibit the proliferation, adhesion, and migration, and increase apoptosis of T cells. In conclusion, the S1P modulator FTY720 could attenuate EAV through the reduction and inhibition of T cells, which might become a novel treatment of ANCA-associated vasculitis.

Urinary mitochondrial DNA is a useful biomarker for assessing kidney injury of antineutrophil cytoplasmic antibody -associated vasculitis.

BACKGROUND: The value of urinary mitochondrial DNA (mtDNA) for assessing kidney injury of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) was investigated. METHODS: Thirty-nine kidney biopsy-proved myeloperoxidase (MPO)-ANCA associated AAV patients were enrolled and analyzed. RESULTS: The average urinary mtDNA of patients was significantly higher than that of normal controls (3372.74 ± 1859.72 vs. 474.90 ± 123.59 copy/nmol creatinine, p < 0.001). The patients who needed dialysis at disease onset had the highest levels of urinary mtDNA (5072.23 ± 1302.87 copy/nmol creatinine). Urinary mtDNA positively correlated with urinary neutrophil gelatinase-associated lipocalin (R = 0.661, P < 0.001) and negatively correlated with estimated glomerular filtration rate (R = -0.515, P = 0.001). The urinary mtDNA level of crescentic class (4703.08 ± 1744.31 copy/nmol creatinine) was higher than that of mixed class (3258.14 ± 1158.99 copy/nmol creatinine) and focal class (2268.15 ± 1897.63 copy/nmol creatinine). Univariate correlation analysis showed urinary mtDNA positively correlated with interstitial neutrophils (R = 0.471, P = 0.048) and glomerular neutrophils (R = 0.673, P = 0.002) in kidney biopsy. Among 13 patients who needed hemodialysis at disease onset, 10 patients who got renal recovery had higher urinary mtDNA than 3 patients who remained dialysis dependent (5455.20 ± 1174.64 vs. 3795.67 ± 893.34 copy/nmol creatinine, p = 0.047). CONCLUSIONS: Urinary mtDNA increases in AAV with kidney injury, and its levels correlate with the severity of kidney injury and neutrophils infiltration in pathology.

Urinary Activin A is a novel biomarker reflecting renal inflammation and tubular damage in ANCA-associated vasculitis.

Activin A, a member of the transforming growth factor-beta superfamily, is a critical modulator of inflammation and plays a key role in controlling the cytokine cascade that drives the inflammatory response. However, the role of activin A in inflammatory kidney diseases remains unknown. To address this issue, we examined here whether activin A can be detected in the kidney and/or urine from patients with antineutrophil cytoplasmic antibody (ANCA) -associated vasculitis (AAV). Fifty-one patients who had been diagnosed with AAV and were treated in our department between November 2011 to March 2018 were included in this study. Forty-one patients had renal complications (renal AAV). Serum and urinary activin A levels were measured by enzyme-linked immunosorbent assay. Correlation of urinary activin A concentration with clinical parameters was analyzed. Urinary activin A was undetectable in healthy volunteers. In contrast, urinary activin A concentration was significantly increased in patients with renal AAV but not in those with non-renal AAV. Urinary activin A concentration decreased rapidly after immunosuppressive treatment. There was a significant correlation of urinary activin A level with urinary protein, L-FABP, and NAG. Histologic evaluation revealed that urinary activin A levels were significantly higher in patients with cellular crescentic glomeruli than in those lacking this damage. In situ hybridization demonstrated that the mRNA encoding the activin A ßA subunit was undetectable in normal kidneys but accumulated in the proximal tubules and crescentic glomeruli of the kidneys of patients with renal AAV. Immunostaining showed that activin A protein also was present in the proximal tubules, crescentic glomeruli, and macrophages infiltrating into the interstitium in the kidneys of patients with renal AAV. These data suggested that urinary activin A concentration reflects renal inflammation and tubular damage in AAV and may be a useful biomarker for monitoring renal AAV.

Clinicopathological analysis of ANCA-associated glomerulonephritis focusing on plasma cell infiltrate.

BACKGROUND: When we encounter glomerulonephritis of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides demonstrating many plasma cell infiltrations, histological overlapping of immunoglobulin G4-related disease (IgG4-RD) often comes into the differential diagnosis. No previous study has focused on the degree of plasma cells in the kidney infiltrate in ANCA-associated glomerulonephritis (ANCA-GN), and the significance of massive plasma cell infiltrate has not been investigated. METHODS: To clarify the plasma cell ratio in renal biopsy specimens of ANCA-GN and the histological characteristic of "plasma cell-rich" ANCA-GN, 20 cases of ANCA-GN were reviewed and clinicopathologically analyzed. RESULTS: Plasma cell ratio was widely distributed between 1.4 and 81%, and the median ratio was 10%. Three patients were categorized in "plasma cell-rich" ANCA-GN, defined as over 45% plasma cell ratio. They tended to include many active glomerular lesions compared to chronic lesions and to display severe tubulointerstitial inflammation. It is suggested that plasma cell-rich ANCA-GN may be acute onset of the disease, and the target of early inflammation may also be in the tubulointerstitial region. Two of the three plasma cell-rich ANCA-GN cases demonstrated numerous IgG4+ cells, but no bird's-eye pattern fibrosis or obliterative phlebitis. CONCLUSIONS: Plasma cell-rich ANCA-GN is not rare and demonstrates distinct clinicopathological characteristics. This study also reminds us that the presence of the significant number of plasma cells in ANCA-GN, as such, is not a histological diagnostic basis for overlap ANCA-GN and IgG4-related disease.

SerpinA3 in the Early Recognition of Acute Kidney Injury to Chronic Kidney Disease (CKD) transition in the rat and its Potentiality in the Recognition of Patients with CKD.

Recognizing patients at early phases of chronic kidney disease (CKD) is difficult, and it is even more challenging to predict acute kidney injury (AKI) and its transition to CKD. The gold standard to timely identify renal fibrosis is the kidney biopsy, an invasive procedure not usually performed for this purpose in clinical practice. SerpinA3 was identified by high-resolution-mass-spectrometry in urines from animals with CKD. An early and progressive elevation of urinary SerpinA3 (uSerpinA3) was observed during the AKI to CKD transition together with SerpinA3 relocation from the cytoplasm to the apical tubular membrane in the rat kidney. uSerpinA3/alpha-1-antichymotrypsin was significantly increased in patients with CKD secondary to focal and segmental glomerulosclerosis (FSGS), ANCA associated vasculitis (AAV) and proliferative class III and IV lupus nephritis (LN). uSerpinA3 levels were independently and positively associated with renal fibrosis. In patients with class V LN, uSerpinA3 levels were not different from healthy volunteers. uSerpinA3 was not found in patients with systemic inflammatory diseases without renal dysfunction. Our observations suggest that uSerpinA3 can detect renal fibrosis and inflammation, with a particular potential for the early detection of AKI to CKD transition and for the differentiation among lupus nephritis classes III/IV and V.

Urinary and serum soluble CD25 complements urinary soluble CD163 to detect active renal anti-neutrophil cytoplasmic autoantibody-associated vasculitis: a cohort study.

Background: Early detection of renal involvement in anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is of major clinical importance to allow prompt initiation of treatment and limit renal damage. Urinary soluble cluster of differentiation 163 (usCD163) has recently been identified as a potential biomarker for active renal vasculitis. However, a significant number of patients with active renal vasculitis test negative using usCD163. We therefore studied whether soluble CD25 (sCD25), a T cell activation marker, could improve the detection of renal flares in AAV. Methods: sCD25 and sCD163 levels in serum and urine were measured by enzyme-linked immunosorbent assay in 72 patients with active renal AAV, 20 with active extrarenal disease, 62 patients in remission and 18 healthy controls. Urinary and blood CD4+ T and CD4+ T effector memory (TEM) cell counts were measured in 22 patients with active renal vasculitis. Receiver operating characteristics (ROC) curves were generated and recursive partitioning was used to calculate whether usCD25 and serum soluble CD25 (ssCD25) add utility to usCD163. Results: usCD25, ssCD25 and usCD163 levels were significantly higher during active renal disease and significantly decreased after induction of remission. A combination of usCD25, usCD163 and ssCD25 outperformed all individual markers (sensitivity 84.7%, specificity 95.1%). Patients positive for sCD25 but negative for usCD163 (n = 10) had significantly higher C-reactive protein levels and significantly lower serum creatinine and proteinuria levels compared with the usCD163-positive patients. usCD25 correlated positively with urinary CD4+ T and CD4+ TEM cell numbers, whereas ssCD25 correlated negatively with circulating CD4+ T and CD4+ TEM cells. Conclusion: Measurement of usCD25 and ssCD25 complements usCD163 in the detection of active renal vasculitis.

Calprotectin as a smoldering activity detection tool and renal prognosis biomarker in ANCA associated vasculitis.

BACKGROUND: Calprotectin is produced by neutrophils and macrophages, and released during the acute phase of the ANCA vasculitis. The aim of our study was to determine if serum and urine calprotectin are disease activity and prognosis biomarkers in ANCA vasculitis patients during remission. METHODS: Forty-two ANCA vasculitis patients were included. Twenty-seven patients were in remission phase under immunosuppressive therapy, and 15 patients were in the acute phase. Four healthy controls were included. We determined calprotectin in serum and urine samples at the time of the inclusion. We recorded the incidence of relapse and the evolution of GFR, proteinuria, hematuria, and C reactive protein and ANCA titer during 24 months of follow-up. RESULTS: In remission phase, serum calprotectin was higher than in healthy controls but lower compared to acute patients (p = 0.05). Serum calprotectin at inclusion was higher in patients who increased proteinuria during follow-up (p = 0.04), with hematuria (p = 0.08), and with non-decreasing ANCA titer (p = 0.0019). Serum calprotectin at inclusion in stable patients who subsequently decreased GFR during follow-up was higher compared with those with a stable or improving GFR (p = 0.03). Urine calprotectin was lower in patients with sclerotic histology in remission (p = 0.03) and acute phase (p = 0.12) compared to the rest of histologies. CONCLUSIONS: Worsening of renal function, hematuria, rising proteinuria and non-decreasing ANCA correlated with higher levels of serum calprotectin at recruitment. Low urine calprotectin was found in patients with sclerotic histology. Calprotectin during remission in ANCA vasculitis may be useful to identify subclinical inflammation and worse renal prognosis patients.

Urinary epidermal growth factor predicts renal prognosis in antineutrophil cytoplasmic antibody-associated vasculitis.

INTRODUCTION: The current study aimed to investigate the association between urinary epidermal growth factor (uEGF) and renal disease severity and outcomes in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV). METHODS: Intrarenal EGFmRNA expression was extracted from transcriptomic data of microdissected tubulointerstitial compartments of kidney biopsies of patients with AAV. uEGF was measured in 173 patients with AAV in active stage and 143 in remission, and normalised to urine creatinine excretion (uEGF/Cr). The association between uEGF/Cr (or EGFmRNA) and clinical-pathological parameters was tested using linear regression analysis. The ability of uEGF/Cr to predict renal outcomes was analysed using Cox's regression analysis. RESULTS: In patients with AAV, intrarenal EGFmRNA expression was significantly associated with estimated glomerular filtration rate (eGFR)(log2) at time of biopsy (ß=0.63, p<0.001). The level of uEGF/Cr was significantly higher in patients in remission than in patients with active disease, both when looking at patients with sequential measurements (2.75±1.03vs 2.08±0.98, p<0.001) and in cross-sectional comparison. uEGF/Cr level was positively associated with eGFR(log2) at time of sampling in both active and remission stage (ß=0.60, p<0.001; ß=0.74, p<0.001, respectively). Patients with resistant renal disease had significantly lower uEGF/Cr levels than responders (1.65±1.22vs 2.16±1.26, p=0.04). Moreover, after adjusting for other potential predictors, uEGF/Cr was independently associated with composite endpoint of end-stage renal disease or 30% reduction of eGFR (HR 0.61, 95% CI 0.45 to 0.83, p=0.001). CONCLUSION: Lower uEGF/Cr levels are associated with more severe renal disease, renal resistance to treatment and higher risk of progression to composite outcome in patients with AAV.

The Utility of Urinalysis in Determining the Risk of Renal Relapse in ANCA-Associated Vasculitis.

BACKGROUND AND OBJECTIVES: The significance of persistent hematuria or proteinuria in patients with ANCA-associated vasculitis who are otherwise in clinical remission is unclear. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A post hoc analysis was conducted using participants enrolled in two randomized, placebo-controlled clinical trials who had active GN due to ANCA-associated vasculitis, had positive ANCA, and achieved remission by month 6. Dipstick and microscopic urinalyses were performed at each visit. Persistent hematuria or proteinuria for at least 6 months and the cumulative duration of hematuria were examined. Renal relapse was defined as new or worsening red blood cell casts and/or worsening kidney function according to the Birmingham Vasculitis Activity Score for Granulomatosis with Polyangiitis. RESULTS: There were 149 patients included in this study: 42% had persistent hematuria, and 43% had persistent proteinuria beyond 6 months. Persistent hematuria was associated with a significantly higher risk of relapse, even after adjusting for potential confounders (subdistribution hazard ratio, 3.99; 95% confidence interval, 1.20 to 13.25; P=0.02); persistent proteinuria was not associated with renal relapse (subdistribution hazard ratio, 1.44; 95% confidence interval, 0.47 to 4.42; P=0.53). Furthermore, greater cumulative duration of hematuria was significantly associated with a higher risk of renal relapse (adjusted subdistribution hazard ratio, 1.08 per each month; 95% confidence interval, 1.03 to 1.12; P<0.01). The median time to renal relapse was 22 months. CONCLUSIONS: In patients with ANCA-associated vasculitis and kidney involvement who achieve remission after induction therapy, the presence of persistent hematuria, but not proteinuria, is a significant predictor of future renal relapse.

[Kidney injury molecules (KIM-1, MCP-1) and type IV collagen in the assessment of activity of antineutrophil cytoplasmic antibody-associated glomerulonephritis]. / Molekuly povrezhdeniia pochechnoi tkani (KIM-1, MCP-1) i kollagen IV tipa v otsenke aktivnosti assotsiirovannogo s antineitrofil'nymi tsitoplazmaticheskimi antitelami glomerulonefrita.

AIM: To assess the significance of determining the serum and urinary concentrations of monocyte chemotactic protein-1 (MCP-1), kidney injury molecule-1 (KIM-1), and type IV collagen in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) to estimate the activity of renal involvement in AAV. SUBJECTS AND METHODS: 78 patients (32 men and 46 women) (median age 55 (45; 61) years) with AAV were examined. The patients were divided into 3 groups according to the AAV activity estimated using the Birmingham vasculitis activity Score (BVAS): 1) 25 patients with active ANCA-associated glomerulonephritis (GN); 2) 26 patients with active AAV without renal involvement; 3) 27 patients in sustained AAV remission. The serum and urinary concentrations of the markers were measured by enzyme immunoassay. RESULTS: The urinary concentration of all 3 biomarkers was higher in patients with renal involvement (Group 1); the differences in the levels of MCP-1 and type IV collagen were statistically significant as compared to Groups 2 and 3 (p<0.01), while that in KIM-1 level was only in Group 2. There were statistically significant correlations between the urinary concentration of these biomarkers and the traditional GN activity indices (erythrocyturia, daily proteinuria (DPU), total BVAS scores that reflect renal involvement, as well as serum creatinine levels and estimated glomerular filtration rate (p<0.05). ROC curve analysis showed that the urinary MCP-1 excretion of ≥159 pg/ml had the highest (92%) sensitivity and urinary type IV collagen excretion of ≥3.09 µg/l had the highest (86%) specificity in assessing the activity of ANCA-associated GN. At the same time, their diagnostic value increased in terms of a combination of DPU and ESR (96% sensitivity, 84.9% specificity). CONCLUSION: The urinary excretion of MCP-1, KIM-1, and type IV collagen reflects the severity of local renal inflammation in AAV patients and a study of these indicators is a promising diagnostic tool for assessing the activity of ANCA-associated GN.

Screening for renal involvement in ANCA-associated vasculitis: room for improvement?

BACKGROUND: Renal involvement in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) requires prompt and aggressive immunosuppressive therapy. The aim of this study was to evaluate screening practice for renal involvement in AAV and its potential effect on renal outcomes. METHODS: Between 2005 and 2015, ANCA-positive AAV patients in a teaching hospital in the Netherlands were retrospectively included. Complete screening for renal involvement was defined as: assessment of erythrocyturia, proteinuria and serum creatinine within two weeks of the diagnosis of AAV. Characteristics at presentation and at 12 months were compared between patients with and without complete screening. RESULTS: A total of 109 AAV patients (63% male) were identified with a mean age of 62 ±; 14 years. Complete screening for renal involvement was performed in 90 of the 109 patients (83%). Patients with incomplete screening had a lower serum creatinine (86 ±; 53 vs. 190 ±; 185 µmol/l, p < 0.001) and were more often diagnosed outside the renal department (100% vs. 78%, p = 0.02). Three patients with incomplete screening had a rise in serum creatinine of ≥ 30% at 12 months. Incomplete screening was not associated with the development of end-stage renal disease. Urine analysis of patients with renal biopsy-proven AAV (n = 31) showed erythrocyturia in 58% after one sample and in 94% after three samples. CONCLUSION: Screening for renal involvement in AAV was suboptimal, primarily in patients who presented outside the renal department. A higher sensitivity for erythrocyturia is achieved if urine analysis is repeated. Incomplete screening may lead to renal impairment if renal involvement is not treated appropriately.

Changes in urinary metabolomic profile during relapsing renal vasculitis.

Current biomarkers of renal disease in systemic vasculitis lack predictive value and are insensitive to early damage. To identify novel biomarkers of renal vasculitis flare, we analysed the longitudinal urinary metabolomic profile of a rat model of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Wistar-Kyoto (WKY) rats were immunised with human myeloperoxidase (MPO). Urine was obtained at regular intervals for 181 days, after which relapse was induced by re-challenge with MPO. Urinary metabolites were assessed in an unbiased fashion using nuclear magnetic resonance (NMR) spectroscopy, and analysed using partial least squares discriminant analysis (PLS-DA) and partial least squares regression (PLS-R). At 56 days post-immunisation, we found that rats with vasculitis had a significantly different urinary metabolite profile than control animals; the observed PLS-DA clusters dissipated between 56 and 181 days, and re-emerged with relapse. The metabolites most altered in rats with active or relapsing vasculitis were trimethylamine N-oxide (TMAO), citrate and 2-oxoglutarate. Myo-inositol was also moderately predictive. The key urine metabolites identified in rats were confirmed in a large cohort of patients using liquid chromatography-mass spectrometry (LC-MS). Hypocitraturia and elevated urinary myo-inositol remained associated with active disease, with the urine myo-inositol:citrate ratio being tightly correlated with active renal vasculitis.

Urinary levels of high mobility group box-1 are associated with disease activity in antineutrophil cytoplasmic autoantibody-associated vasculitis.

BACKGROUND: High mobility group box-1 (HMGB1), a kind of pro-inflammatory mediator, is associated with inflammatory conditions and tissue damage. Our previous study demonstrated that the circulating levels of HMGB1 correlated with disease activity of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In the current study, we aimed to measure urinary levels of HMGB1 in AAV patients, correlated them to clinical activity index and analysed the immunohistochemical HMGB1 staining in kidney specimens. METHODS: 50 patients with AAV in active stage and 56 patients with AAV in remission were recruited. The urinary levels of HMGB1 were determined by enzyme-linked immunosorbent assay. Moreover, renal biopsy specimens from 27 patients with active AAV were randomly collected to evaluate the deposition of HMGB1. RESULTS: Urinary HMGB1 levels in AAV patients in active stage were significantly higher than those in AAV patients in remission and healthy controls (1.46 [0.56-3.43] versus 0.38 [0.10-1.35] mg/µmolCr, P=0.001; 1.46 [0.56-3.43] versus 0.48 [0.40-0.60] mg/µmolCr, P=0.000, respectively). Further analysis found that urinary levels of HMGB1 correlated with erythrocyte sedimentation rate (r=0.354, p=0.012), C-reactive protein (r=0.289, p=0.042), and Birmingham Vasculitis Activity Score (r=0.350, p=0.013). Renal tissue of active AAV patients showed HMGB1 was mainly expressed in the cytoplasm and the extracellular space. The percentage of HMGB1-negative nuclei in renal tissue of patients with active AAV was significantly higher than that in normal controls (60.6±20.2 % versus 2.7±0.6 %, p<0.01). CONCLUSION: Urinary levels of HMGB1 may be associated with the disease activity in AAV patients.