Longitudinal assessment of urinary ALCAM, HPX, and PRDX6 in Korean patients with systemic lupus erythematosus: implications for disease activity monitoring and treatment response.

Introduction: This study aimed to demonstrate the potential of activated leukocyte cell adhesion molecule (ALCAM), hemopexin (HPX), and peroxiredoxin 6 (PRDX6) as urine biomarkers for systemic lupus erythematosus (SLE). Methods: Urine samples were collected from 138 Korean patients with SLE from the Ajou Lupus Cohort and 39 healthy controls (HC). The concentrations of urine biomarkers were analyzed using enzyme-linked immunosorbent assay kits specific for ALCAM, HPX, and PRDX6, respectively. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic utility, and Pearson's correlation analysis was conducted to assess the relationships between the disease activity and urine biomarkers. Results: Patients with SLE and patients with lupus nephritis (LN) showed significantly elevated ALCAM, HPX, and PRDX6 levels compared with HCs. ALCAM, HPX, and PRDX6 showed significant diagnostic values, especially for lupus nephritis (LN), with areas under the receiver operating characteristic curve for LN was 0.850 for ALCAM (95% CI, 0.778-0.921), 0.781 for HPX (95% CI, 0.695-0.867), and 0.714 for PRDX6 (95% CI, 0.617-0.812). Correlation analysis revealed that all proteins were significantly associated with anti-double stranded DNA antibody (ALCAM, r = 0.350, p < 0.001; HPX, r = 0.346, p < 0.001; PRDX6, r = 0.191, p = 0.026) and SLEDAI (ALCAM, r = 0.526, p < 0.001; HPX, r = 0.479, p < 0.001; PRDX6, r = 0.262, p = 0.002). Results from the follow-up of the three biomarker levels in these patients revealed a significant decrease, showing a positive correlation with changes in SLEDAI-2k scores (ALCAM, r = 0.502, p < 0.001; HPX, r = 0.475, p < 0.001; PRDX6, r = 0.245, p = 0.026), indicating their potential as indicators for tracking disease activity. Discussions: Urinary ALCAM, HPX, and PRDX6 levels have diagnostic value and reflect disease activity in Korean patients with SLE, emphasizing their potential for non-invasive monitoring and treatment response evaluation.

Urinary soluble CD163 is a putative non-invasive biomarker for primary sclerosing cholangitis.

Soluble CD163 (sCD163) is a selective marker of macrophages whose circulating levels have been found to be induced in patients with active inflammatory bowel disease (IBD). Urinary proteins are emerging as non-invasive diagnostic biomarkers, and here, sCD163 levels were measured in the urine of 18 controls and 63 patients with IBD by enzyme-linked immunosorbent assay. Urinary sCD163 levels did, however, not differentiate IBD patients from controls. Analysis of sCD163 in the serum of 51 of these patients did not show higher levels in IBD. Primary sclerosing cholangitis (PSC) is often associated with IBD, and sCD163 was higher in the urine of the 21 patients and in the serum of the 13 patients with PSC compared to patients with IBD. Of clinical relevance, urinary sCD163 levels were higher in PSC patients compared to those with other chronic liver diseases (n = 16), while serum sCD163 levels were comparable between the two groups. Serum sCD163 of IBD and PSC patients positively correlated with serum C-reactive protein. Serum creatinine and glomerular filtration rate, surrogate markers for renal function, did not significantly correlate with urinary or serum sCD163 levels in IBD or PSC patients. Moreover, urinary sCD163 was not related to fecal calprotectin levels whereas serum sCD163 of IBD patients showed a positive trend. PSC associated with IBD and PSC without underlying IBD had similar levels of urinary sCD163 while serum sCD163 tended to be higher in the latter group. In PSC patients, urinary sCD163 did not correlate with serum aminotransferase levels, gamma glutamyl transferase, alkaline phosphatase, bilirubin or the Model for End Stage Liver Disease score. Ursodeoxycholic acid was prescribed to our PSC patients and fecal levels of ursodeoxycholic acid and its conjugated forms were increased in PSC compared to IBD patients. Otherwise, fecal bile acid levels of IBD and PSC patients were almost identical, and were not correlated with urinary and serum sCD163 in PSC. In summary, our study identified urinary sCD163 as a potential biomarker for PSC.

Longitudinal urinary biomarkers of immunological activation in covid-19 patients without clinically apparent kidney disease versus acute and chronic failure.

Kidney function is affected in COVID-19, while kidney itself modulates the immune response. Here, hypothesize if COVID-19 urine biomarkers level can assess immune activation vs. clinical trajectory. Considering the kidney's critical role in modulating the immune response, we sought to analyze activation markers in patients with pre-existing dysfunction. This was a cross-sectional study of 68 patients. Blood and urine were collected within 48 h of hospital admission (H1), followed by 96 h (H2), seven days (H3), and up to 25 days (H4) from admission. Serum level ferritin, procalcitonin, IL-6 assessed immune activation overall, while the response to viral burden was gauged with serum level of spike protein and αspike IgM and IgG. 39 markers correlated highly between urine and blood. Age and race, and to a lesser extend gender, differentiated several urine markers. The burden of pre-existing conditions correlated with urine DCN, CAIX and PTN, but inversely with IL-5 or MCP-4. Higher urinary IL-12 and lower CAIX, CCL23, IL-15, IL-18, MCP-1, MCP-3, MUC-16, PD-L1, TNFRS12A, and TNFRS21 signified non-survivors. APACHE correlated with urine TNFRS12, PGF, CAIX, DCN, CXCL6, and EGF. Admission urine LAG-3 and IL-2 predicted death. Pre-existing kidney disease had a unique pattern of urinary inflammatory markers. Acute kidney injury was associated, and to a certain degree, predicted by IFNg, TWEAK, MMP7, and MUC-16. Remdesavir had a more profound effect on the urine biomarkers than steroids. Urinary biomarkers correlated with clinical status, kidney function, markers of the immune system activation, and probability of demise in COVID-19.

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.

Excretion of urine extracellular vesicles bearing markers of activated immune cells and calcium/phosphorus physiology differ between calcium kidney stone formers and non-stone formers.

BACKGROUNDS: Previous studies have demonstrated that excretion of urinary extracellular vesicles (EVs) from different nephron segments differs between kidney stone formers and non-stone formers (NSFs), and could reflect pathogenic mechanisms of urinary stone disease. In this study we quantified selected populations of specific urinary EVs carrying protein markers of immune cells and calcium/phosphorus physiology in calcium oxalate stone formers (CSFs) compared to non-stone formers (NSFs). METHODS: Biobanked urine samples from CSFs (n = 24) undergoing stone removal surgery and age- and sex- matched NSFs (n = 21) were studied. Urinary EVs carrying proteins related to renal calcium/phosphorus physiology (phosphorus transporters (PiT1 and PiT2), Klotho, and fibroblast growth factor 23 (FGF23); markers associated with EV generation (anoctamin-4 (ANO4) and Huntington interacting protein 1 (HIP1)), and markers shed from activated immune cells were quantified by standardized and published method of digital flow cytometry. RESULTS: Urine excretion of calcium, oxalate, phosphorus, and calcium oxalate supersaturation (SS) were significantly higher in CSFs compared to NSFs (P < 0.05). Urinary excretion of EVs with markers of total leukocytes (CD45), neutrophils (CD15), macrophages (CD68), Klotho, FGF23, PiT1, PiT2, and ANO4 were each markedly lower in CSFs than NSFs (P < 0.05) whereas excretion of those with markers of monocytes (CD14), T-Lymphocytes (CD3), B-Lymphocytes (CD19), plasma cells (CD138 plus CD319 positive) were not different between the groups. Urinary excretion of EVs expressing PiT1 and PiT2 negatively (P < 0.05) correlated with urinary phosphorus excretion, whereas excretion of EVs expressing FGF23 negatively (P < 0.05) correlated with both urinary calcium and phosphorus excretion. Urinary EVs with markers of HIP1 and ANO4 correlated negatively (P < 0.05) with clinical stone events and basement membrane calcifications on papillary tip biopsies. CONCLUSIONS: Urinary excretion of EVs derived from specific types of activated immune cells and EVs with proteins related to calcium/phosphorus regulation differed between CSFs and NSFs. Further validation of these and other populations of urinary EVs in larger cohort could identify biomarkers that elucidate novel pathogenic mechanisms of calcium stone formation in specific subsets of patients.

Increased Urinary CD163 Levels in Systemic Vasculitis with Renal Involvement.

OBJECTIVES: Systemic vasculitis includes a group of disorders characterized by inflammation of the vessel wall, involving multiple systems, and can cause malignant hypertension. CD163 is a specific marker of anti-inflammatory macrophages. This study is aimed at evaluating the CD163 levels in relation to systemic vasculitis and renal involvements. METHODS: Urinary CD163 levels were retrospectively measured by enzyme-linked immunosorbent assay (ELISA) in 51 patients with systemic vasculitis, 42 essential hypertensions, and 36 healthy volunteers. The associations between urinary CD163 levels and clinical indicators were analyzed. RESULTS: Urinary CD163 levels were significantly higher in patients with systemic vasculitis [68.20 (38.25~158.78) (pg/ml)] compared to essential hypertension [43.86 (23.30-60.71) (pg/ml)] (p = 0.003) and the healthy volunteers [30.76 (9.30-54.16) (pg/ml)] (p < 0.001). Furthermore, systemic vasculitis patients with renal involvement had significantly higher urinary CD163 levels relative to patients without renal involvement [86.95 (47.61 and 192.38) pg/ml] vs. [41.99 (17.70 and 71.95) pg/ml, p = 0.005]. After control factors age, sex, and BMI, urinary CD163 levels in systemic vasculitis patients were positively correlated with serum creatinine, blood urea nitrogen, and ß-2 microglobulin (r = 0.45, 0.48, and 0.46; p = 0.001, 0.001, and 0.002, respectively). In addition, we found the level of urinary CD163 in granulomatous vasculitis (including TA, GPA, and EGPA) was significantly higher than that in necrotizing vasculitis (including PAN) [86.95 (41.99 and 184.82) pg/ml] vs. [45.73 (21.43 and 74.43) pg/ml, p = 0.016]. CONCLUSION: Urinary CD163 levels were significantly higher in patients with systemic vasculitis, especially in patients with renal involvement. Thus, urinary CD163 has the potential to be a biomarker for systemic vasculitis with renal involvement.

Urinary Soluble CD163 Levels Predict IgA Nephropathy Remission Status.

Noninvasive biomarkers of disease activity are needed to predict disease remission status in patients with IgA nephropathy (IgAN). Soluble CD163 (sCD163), shed by monocytes and macrophages, is a potential biomarker in diseases associated with excessive macrophage activation. We investigated the association of urinary sCD163 (u-sCD163) with histopathological activity and clinical manifestations in 349 patients with biopsy-diagnosed IgAN. U-sCD163 was measured via enzyme-linked immunosorbent assay. In patients with IgAN, higher u-sCD163 levels were associated with histological lesions of greater severity, as well as more proteinuria and poorer renal function. Additionally, u-sCD163 was correlated with infiltration of tubulointerstitial CD163+ macrophages. High u-sCD163 levels (>3.57 ng/mg Cr) were associated with a 2.66-fold greater risk for IgAN remission failure in adjusted analyses. Adding u-sCD163 levels to the model containing clinical data at biopsy and MEST-C score significantly improved the risk prediction of IgAN remission status (AUC 0.788). Together, our results suggest that u-sCD163 may be a useful noninvasive biomarker to evaluate disease severity and remission status of IgAN.

Clinical impact of urinary CD11b and CD163 on the renal outcomes of anti-neutrophil cytoplasmic antibody-associated glomerulonephritis.

BACKGROUND: The detection of leukocyte-derived CD11b (α subunit of integrin Mac-1) and CD163 (scavenger receptor) in urine may reflect renal inflammation in antineutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN). The objective of this study was to evaluate the clinical significance of urinary CD11b (U-CD11b) and CD163 (U-CD163) in ANCA-GN. METHODS: U-CD11b and U-CD163 were examined using enzyme-linked immunosorbent assay in ANCA-GN urine samples from our institutional cohort (n = 88) and a nationwide cohort (n = 138), and their association with renal histology was subsequently analyzed. Logistic regression analyses were performed on a nationwide ANCA cohort to determine the associations of the two urinary molecules with renal remission failure at 6 months or with yearly estimated glomerular filtration rate (eGFR) slope over a 24-month observation period. RESULTS: U-CD11b and U-CD163 were significantly associated with cellular crescent formation and leukocyte accumulation in glomerular crescents. With regard to interstitial inflammation, both levels of U-CD11b and U-CD163 at diagnosis remarkably increased in ANCA-GN compared with the levels observed in nonglomerular kidney disorders including nephrosclerosis, immunoglobulin G4-related disease and tubulointerstitial nephritis; however, the presence of U-CD11b alone was significantly correlated with tubulointerstitial leukocyte infiltrates. Although neither U-CD11b nor U-CD163 at diagnosis was associated with remission failure at 6 months, multivariate analysis demonstrated that the baseline U-CD11b levels were significantly associated with the increase in eGFR following immunosuppressive therapy. CONCLUSIONS: Although both U-CD11b and U-CD163 reflect renal leukocyte accumulation, U-CD11b at diagnosis provides additional clinical value by predicting the recovery rate after the treatment of ANCA-GN.

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.

Association of Urine sCD163 With Proliferative Lupus Nephritis, Fibrinoid Necrosis, Cellular Crescents and Intrarenal M2 Macrophages.

CD163 is a marker for alternatively activated macrophages, which have been implicated in the pathogenesis of lupus nephritis (LN). In our preliminary screening of urine proteins in LN, urine soluble CD163 (sCD163) was significantly elevated in patients with active LN. To evaluate the potential of sCD163 as a biomarker in LN, urine sCD163 was assayed in patients with active LN, active non-renal lupus patients (ANR), inactive SLE and healthy controls (HC), using ELISA and normalized to urine creatinine. The correlation of urine sCD163 with clinical parameters and renal pathological attributes was further investigated in LN patients with concurrent renal biopsies. A total of 228 SLE patients and 56 HC were included from three cohorts. Results demonstrated that urine sCD163 was significantly elevated in active LN when compared with HC, inactive SLE, or ANR in African-American, Caucasian and Asian subjects (all P < 0.001). In LN patients with concurrent renal biopsies, urine sCD163 was significantly increased in patients with proliferative LN when compared with non-proliferative LN (P < 0.001). Urine sCD163 strongly correlated with SLEDAI, rSLEDAI, activity index (AI) of renal pathology, fibrinoid necrosis, cellular crescents, and interstitial inflammation on biopsies (all P < 0.01). Macrophages, particularly M2 macrophages, the predominant cells expressing CD163 within LN kidneys, represented a potential source of elevated urine sCD163, based on single-cell RNA sequencing analysis. To conclude, urine sCD163 discriminated patients with active LN from other SLE patients and was significantly elevated in proliferative LN. It strongly correlated with concurrent AI and several specific pathological attributes, demonstrating its potential in predicting renal pathology.

Urinary Soluble CD163: a Novel Noninvasive Biomarker of Activity for Lupus Nephritis.

BACKGROUND: Clinical distinction between patients with lupus nephritis who have active inflammation or chronic kidney damage is challenging. Studies have shown soluble CD163, which derives from cleavage of the CD163 M2c macrophage receptor and can be quantified in urine, correlates with active lupus nephritis. METHODS: We measured urine CD163 at lupus nephritis flares in patients from a Mexican cohort and cross-sectional and longitudinal United States cohorts. We also performed serial urine CD163 measurements during the treatment of flares in a subset of patients from the Mexican and longitudinal United States cohorts, and assessed response to therapy at 12 months. In addition, we evaluated urinary CD163 agreement with histologic activity in 19 patients from the Mexican cohort who had repeated kidney biopsies on follow-up. RESULTS: Urinary CD163 levels were significantly higher in patients with active lupus nephritis than in patients with active extrarenal SLE, inactive SLE, and other glomerular diseases, and correlated with disease clinical severity, histologic class, and the histologic activity index. Urinary CD163 increased from 6 months preflare to flare, diminishing progressively in complete and partial responders, whereas it remained elevated in nonresponders. Urinary CD163 <370 ng/mmol at 6 months predicted complete renal response at 12 months with >87% sensitivity and >87% specificity. Urinary CD163 <370 ng/mmol or >370 ng/mmol perfectly agreed (κ=1.0) with a histologic activity index ≤1 or >1 in repeated biopsies, respectively. Evaluation of urinary CD163 in patients with persistent proteinuria at 6 months improved the prediction of who would achieve complete renal response at 12 months. CONCLUSIONS: Urinary CD163 reflects histologic inflammation in lupus nephritis and is a promising activity biomarker that varies over time with lupus nephritis activity and treatment.

Urinary soluble CD163 and monocyte chemoattractant protein-1 in the identification of subtle renal flare in anti-neutrophil cytoplasmic antibody-associated vasculitis.

BACKGROUND: Prior work has shown that urinary soluble CD163 (usCD163) displays excellent biomarker characteristics for detection of active renal vasculitis using samples that included new diagnoses with highly active renal disease. This study focused on the use of usCD163 in the detection of the more clinically relevant state of mild renal flare and compared results of usCD163 testing directly to testing of urinary monocyte chemoattractant protein-1 (uMCP-1). METHODS: Patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV, n = 88) were identified within a serially sampled, longitudinal and multicentre cohort. Creatinine-normalized usCD163 and uMCP-1 levels were measured by enzyme-linked immunosorbent assay and, both alone and in combination, were compared between times of active renal AAV and during remission and/or active non-renal AAV. RESULTS: Samples from 320 study visits included times of active renal vasculitis (n = 39), remission (n = 233) and active extrarenal vasculitis (n = 48). Median creatinine levels were 0.9 mg/dL [interquartile range (IQR) 0.8-1.2] in remission and 1.4 mg/dL (IQR 1.0-1.8) during renal flare. usCD163 levels were higher in patients with active renal vasculitis compared with patients in remission and those with active extrarenal vasculitis, with median values of 162 ng/mmol (IQR 79-337), 44 (17-104) and 38 (7-76), respectively (P < 0.001). uMCP-1 levels were also higher in patients with active renal vasculitis compared with patients in remission and those with active extrarenal vasculitis, with median values of 10.6 pg/mmol (IQR 4.6-23.5), 4.1 (2.5-8.4) and 4.1 (1.9-6.8), respectively (P < 0.001). The proposed diagnostic cut-points for usCD163 and uMCP-1 were 72.9 ng/mmol and 10.0 pg/mmol, respectively. usCD163 and uMCP-1 levels were marginally correlated (r2 = 0.11, P < 0.001). Combining novel and existing biomarkers using recursive tree partitioning indicated that elevated usCD163 plus either elevated uMCP-1 or new/worse proteinuria improved the positive likelihood ratio (PLR) of active renal vasculitis to 19.2. CONCLUSION: A combination of usCD163 and uMCP-1 measurements appears to be useful in identifying the diagnosis of subtle renal vasculitis flare.

ALCAM and VCAM-1 as urine biomarkers of activity and long-term renal outcome in systemic lupus erythematosus.

OBJECTIVES: We investigated the cell adhesion molecules (CAMs) Vascular CAM 1 (VCAM-1) and Activated Leucocyte CAM (ALCAM) as urinary biomarkers in SLE patients with and without renal involvement. METHODS: Female SLE patients (n = 111) and non-SLE population-based controls (n = 99) were enrolled. We measured renal activity using the renal domain of the BILAG index and urine (U) and plasma (P) concentrations of soluble (s)VCAM 1 and U-sALCAM using ELISA. U-sCAM levels were next corrected by U-creatinine. RESULTS: U-sVCAM-1/creatinine and U-sALCAM/creatinine ratios were higher in SLE patients vs non-SLE controls (P < 0.001 for both), as well as in patients with active/low-active (BILAG A-C; n = 11) vs quiescent (BILAG D; n = 19) LN (P = 0.023 and P = 0.001, respectively). U-sALCAM/creatinine but not U-sVCAM-1/creatinine ratios were higher in patients with nephritis history (BILAG A-D; n = 30) vs non-renal SLE (BILAG E; n = 79) (P = 0.014). Patients with baseline U-sVCAM-1/creatinine ratios ≥75th percentile showed a 23-fold increased risk of a deterioration in estimated glomerular filtration rate by ≥25% during a 10-year follow-up (odds ratio: 22.9; 95% CI: 2.8, 189.2; P = 0.004); this association remained significant after adjustments for age, disease duration and organ damage. Traditional markers including anti-dsDNA antibodies did not predict this outcome. CONCLUSION: While high U-sVCAM-1 levels appear to reflect SLE disease activity, sALCAM might have particular importance in renal SLE. Both U-sVCAM-1 and U-sALCAM showed ability to distinguish SLE patients with active renal involvement from patients with quiescent or no prior nephritis. High U-sVCAM-1 levels may indicate patients at increased risk for long-term renal function loss.

Urine Angiotensin II Signature Proteins as Markers of Fibrosis in Kidney Transplant Recipients.

BACKGROUND: Interstitial fibrosis/tubular atrophy (IFTA) is an important cause of kidney allograft loss; however, noninvasive markers to identify IFTA or guide antifibrotic therapy are lacking. Using angiotensin II (AngII) as the prototypical inducer of IFTA, we previously identified 83 AngII-regulated proteins in vitro. We developed mass spectrometry-based assays for quantification of 6 AngII signature proteins (bone marrow stromal cell antigen 1, glutamine synthetase [GLNA], laminin subunit beta-2, lysophospholipase I, ras homolog family member B, and thrombospondin-I [TSP1]) and hypothesized that their urine excretion will correlate with IFTA in kidney transplant patients. METHODS: Urine excretion of 6 AngII-regulated proteins was quantified using selected reaction monitoring and normalized by urine creatinine. Immunohistochemistry was used to assess protein expression of TSP1 and GLNA in kidney biopsies. RESULTS: The urine excretion rates of AngII-regulated proteins were found to be increased in 15 kidney transplant recipients with IFTA compared with 20 matched controls with no IFTA (mean log2[fmol/µmol of creatinine], bone marrow stromal cell antigen 1: 3.8 versus 3.0, P = 0.03; GLNA: 1.2 versus -0.4, P = 0.03; laminin subunit beta-2: 6.1 versus 5.4, P = 0.06; lysophospholipase I: 2.1 versus 0.6, P = 0.002; ras homolog family member B: 1.2 versus -0.1, P = 0.006; TSP1_GGV: 2.5 versus 1.9; P = 0.15; and TSP1_TIV: 2.0 versus 0.6, P = 0.0006). Receiver operating characteristic curve analysis demonstrated an area under the curve = 0.86 for the ability of urine AngII signature proteins to discriminate IFTA from controls. Urine excretion of AngII signature proteins correlated strongly with chronic IFTA and total inflammation. In a separate cohort of 19 kidney transplant recipients, the urine excretion of these 6 proteins was significantly lower following therapy with AngII inhibitors (P < 0.05). CONCLUSIONS: AngII-regulated proteins may represent markers of IFTA and guide antifibrotic therapies.

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.

Soluble CD163 as a Potential Biomarker in Systemic Sclerosis.

OBJECTIVE: To evaluate the performance of serum and urinary sCD163 concentrations as possible biomarker in systemic sclerosis (SSc). METHODS: Urine and serum samples were obtained from SSc patients and age- and sex-matched controls. Serum and urinary sCD163 concentrations were measured by commercially available ELISA kit. SSc patients were assessed following international guidelines. Cross-sectional analyses were performed. RESULTS: Two hundred and three SSc patients were included. The control group consisted of 47 age- and sex-matched patients having noninflammatory diseases, mainly osteoporosis. Serum sCD163 levels were significantly higher in SSc patients compared with controls (mean ± SD: 529 ± 251 versus 385 ± 153 ng/mL; p < 0.001). Urinary sCD163 concentrations were higher in SSc patients than controls, but this did not reach significance (236 ± 498 versus 176 ± 173 ng/mg uCr; p = 0.580). The sCD163 concentrations were not associated with clinical, laboratory, and instrumental characteristics of SSc patients. CONCLUSION: To our knowledge, this is the first evaluation of both serum and urinary sCD163 levels in SSc. Our results show a significant difference for sera values that should be prioritized for further studies as compared to urinary measurements. Our results further support that the M2 macrophages/CD163 signaling system may play a role in the pathogenesis of SSc, although we could not identify a subset of SSc patients with higher concentrations.

Biomarkers in vasculitis.

PURPOSE OF REVIEW: Biomarkers are considered to be helpful in diagnosing, monitoring, predicting treatment response, and prognosis in clinical practice and as outcomes in clinical trials. In this article, we review the recent literature on new biomarkers and the expanding use of older ones in vasculitic conditions. RECENT FINDINGS: In antineutrophil cytoplasmic antibody-associated vasculitis patients antineutrophil cytoplasmic antibody type may be useful as a predictor of relapse and response to rituximab. Moreover, serial measurements of proteinase-3 titer may help to predict relapse. Urinary soluble CD163 levels are promising for identifying active renal vasculitis. Imaging modalities such as positron emission tomography, computerized angiography tomography, and temporal artery ultrasound maintain their role in diagnosis and disease assessment in large vessel vasculitis. Fecal calprotectin is a useful marker of active gastrointestinal involvement in Behçet's syndrome. SUMMARY: The publications reviewed here potentially may help to move the field of biomarkers in vasculitis management. However, more work toward understanding the underlying pathophysiology and effects of an intervention on the disease process are needed before true biomarkers can be realized. Further studies with appropriate control groups, using good definitions for disease states such as activity and remission are needed to guide our use of these markers correctly in the management of our patients.

Separation and Analysis of Lactosylceramide, Galabiosylceramide, and Globotriaosylceramide by LC-MS/MS in Urine of Fabry Disease Patients.

Fabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase A (α-GAL A) deficiency. This enzyme contributes to the cellular recycling of glycosphingolipids such as galabiosylceramide (Ga2), globotriaosylceramide (Gb3), and globotriaosylsphingosine (lyso-Gb3) by hydrolyzing the terminal α-galactosyl moiety. Urine and plasma α-GAL A substrates are currently analyzed as biomarkers for the detection, monitoring, and follow-up of Fabry disease patients. The sensitivity of the analysis of Ga2 is decreased by the co-analysis of its structural isomer, lactosylceramide (LacCer), which is not an α-GAL A substrate. A normal-phase ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) methodology, allowing the baseline separation of 12 Ga2 isoforms/analogues from their lactosylceramide counterparts, was developed and validated in urine. The method was multiplexed with the analysis of 12 Gb3 isoforms/analogues having the same fatty acid moieties as those of Ga2 for comparison, and with creatinine for sample normalization. Urine samples were studied from 34 untreated and 33 Fabry males treated by enzyme replacement therapy (ERT) and 54 untreated and 19 ERT-treated Fabry females, along with 34 male and 25 female healthy controls. The chromatographic separation of Ga2 from LacCer increased the sensitivity of analysis, especially in women. One untreated Fabry female and two treated Fabry females presented abnormal levels of Ga2 but normal levels of Gb3, supporting the importance of analyzing Ga2, in addition to Gb3. Our results show that urine LacCer levels from females were significantly higher than those from males. Moreover, LacCer levels were not affected by Fabry disease for both males and females.

Urinary soluble CD163 level reflects glomerular inflammation in human lupus nephritis.

BACKGROUND: In addition to classically activated macrophages that have effector roles in tissue injury, alternatively activated M2 macrophages are involved in the resolution of inflammation in animal models of kidney disease. To clarify the clinical relevance of macrophage phenotypes in human glomerular diseases, we evaluated the renal accumulation of macrophages and plasma and urine levels of CD163, an M2 marker, in lupus nephritis (LN) patients. METHODS: Kidney biopsies and plasma and urine samples were obtained from LN patients who underwent renal biopsy between 2008 and 2012. CD163+, CD68+ and CD204+ cells were counted in paraffin-embedded and frozen sections. LN histological activity was evaluated semiquantitatively using the biopsy activity index. Plasma and urinary soluble CD163 (sCD163) concentrations were also measured and evaluated for their significance as potential LN biomarkers. RESULTS: Immunohistological analysis of glomeruli from LN patients revealed that >60% of CD68+ macrophages had merged with CD163+ cells. The increased number of glomerular CD163+ macrophages was correlated with LN severity, as determined by the biopsy active index (r = 0.635). Urinary (u-) sCD163 level was strongly correlated with glomerular CD163+ cell counts and histological disease score as well as urinary monocyte chemoattractant protein 1 levels (r = 0.638 and 0.592, respectively). Furthermore, the u-sCD163 level was higher in patients with active LN than in those with other diseases. CONCLUSIONS: Glomerular CD163+ macrophages are the predominant phenotype in the kidneys of lupus patients. These findings indicate that the u-sCD163 level can serve as a biomarker for macrophage-dependent glomerular inflammation in human LN.

Urinary Soluble CD163 in Active Renal Vasculitis.

A specific biomarker that can separate active renal vasculitis from other causes of renal dysfunction is lacking, with a kidney biopsy often being required. Soluble CD163 (sCD163), shed by monocytes and macrophages, has been reported as a potential biomarker in diseases associated with excessive macrophage activation. Thus, we hypothesized that urinary sCD163 shed by crescent macrophages correlates with active glomerular inflammation. We detected sCD163 in rat urine early in the disease course of experimental vasculitis. Moreover, microdissected glomeruli from patients with small vessel vasculitis (SVV) had markedly higher levels of CD163 mRNA than did those from patients with lupus nephritis, diabetic nephropathy, or nephrotic syndrome. Both glomeruli and interstitium of patients with SVV strongly expressed CD163 protein. In 479 individuals, including patients with SVV, disease controls, and healthy controls, serum levels of sCD163 did not differ between the groups. However, in an inception cohort, including 177 patients with SVV, patients with active renal vasculitis had markedly higher urinary sCD163 levels than did patients in remission, disease controls, or healthy controls. Analyses in both internal and external validation cohorts confirmed these results. Setting a derived optimum cutoff for urinary sCD163 of 0.3 ng/mmol creatinine for detection of active renal vasculitis resulted in a sensitivity of 83%, specificity of 96%, and a positive likelihood ratio of 20.8. These data indicate that urinary sCD163 level associates very tightly with active renal vasculitis, and assessing this level may be a noninvasive method for diagnosing renal flare in the setting of a known diagnosis of SVV.