Urinary angiotensinogen: an indicator of active antineutrophil cytoplasmic antibody-associated glomerulonephritis

Urinary angiotensinogen: an indicator of active antineutrophil cytoplasmic antibody-associated... Abstract Background One of the major challenges in improving the management of antineutrophil cytoplasmic antibody–associated glomerulonephritis (ANCA-GN) is the lack of a disease-specific indicator for histological lesions and disease activity. Here we tested the utility of urinary angiotensinogen (UAGT) as a biomarker of renal disease activity in ANCA-GN. Methods A prospective, two-stage cohort study was performed in ANCA-GN patients. In Stage I, UAGT was measured at the time of renal biopsy in 69 patients from two centers (test set) and 25 patients from two other centers (validation set). In Stage II, UAGT was monitored in 50 subjects in the test set for 24 months. Results In Stage I, UAGT significantly increased in ANCA-GN patients, correlating well with cellular crescents formation and active interstitial inflammation. Patients with crescentic ANCA-GN exhibited the highest UAGT compared with other histopathological classes of ANCA-GN. After multivariable adjustment, the highest quartile of UAGT, compared with the lowest quartile, associated with a 6-fold increased risk of crescentic ANCA-GN. For predicting crescentic ANCA-GN, UAGT [area under the receiver operating characteristics curve (AUC) = 0.88] outperformed albuminuria (AUC = 0.73) and estimated glomerular filtration rate (AUC = 0.69). UAGT improved the performance of those clinical markers in diagnosing crescentic ANCA-GN (P < 0.034), suggesting a role of UAGT in identifying active crescentic ANCA-GN. In Stage II, UAGT decreased after immunotherapy and increased at the time of renal relapse during the 2-year follow-up, suggesting the usefulness of UAGT to monitor disease activity over time. Conclusions These results suggest the potential use of UAGT for assessing disease activity and renal relapse in ANCA-GN. ANCA glomerulonephritis, disease activity, indicator, renal pathology, urinary angiotensinogen INTRODUCTION Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is the leading cause of rapidly progressive crescentic glomerulonephritis (GN) [1, 2]. Current therapeutic strategies have substantial side effects and relapses are frequent [3–5]. The mortality of patients in the first year of therapy is from adverse events rather than from the disease itself [6]. Investigations that can be used to detect renal disease activity and treatment response are helpful in optimizing therapy and minimizing treatment-associated side effects. The widespread use of ANCA assays facilitates the diagnosis of ANCA-GN, but the ANCA level does not correlate well with renal disease activity [7]. Assessments of renal disease activity are only achievable with renal biopsies, which are obtained from patients via an invasive operation [2, 8]. In this setting, development of a noninvasive surrogate biomarker that closely parallels renal pathological activity is urgently needed. The pathogenesis of ANCA-GN is multifactorial but is likely related to necrotizing vascular inflammation as well as crescents formation in glomeruli [9, 10]. There is substantial evidence that activation of the intrarenal renin–angiotensin system (RAS) drives leukocyte recruitment and renal cell proliferation and thereby plays an important role in the development of proliferative and crescentic GN [11–13]. Indeed, enhanced intrarenal production of angiotensin II and angiotensinogen (AGT) has been observed in animals with crescentic GN [12, 14, 15]. Treatment with RAS blockers markedly ameliorates crescentic lesions in GN [12, 16, 17]. Furthermore, AGT, an important substrate of angiotensin II, can be secreted from AGT-positive cells in the kidney and be detectable in the urine [11]. Urinary AGT (UAGT) has been proposed as a biomarker of renal disease severity [18, 19] and has been shown to associate with renal outcome in certain diseases [19–23]. However, the exact levels of UAGT and their relevance to renal pathology and disease activity in human ANCA-GN remain to be elucidated. We conducted a prospective observational study in 69 patients with biopsy-proven ANCA-GN. Our objective is to test and validate the hypothesis that UAGT could serve as a useful biomarker for renal pathology activity and could be used to monitor treatment response and renal relapse. This is the first study in ANCA-GN to demonstrate the utility of UAGT as an indicator of disease activity and renal relapse. MATERIALS AND METHODS Patients This study was performed in accordance with the Declaration of Helsinki and was approved by the institutional ethical review board for each center. All the participants provided written informed consent. Sample collection for the test set was conducted in two centers in Wenzhou and Guangzhou from September 2012 to August 2017. The validation test was conducted in two other centers in Guangzhou from January 2016 to March 2018. Patients with biopsy-proven ANCA-GN in the two sets were enrolled according to the inclusion and exclusion criteria described below. Eligible participants were patients with biopsy-proven ANCA-GN ages 18–80 years. Exclusion criteria include those with inadequate biopsy specimens (<10 glomeruli), those being treated with RAS inhibitors at the time of sample collection, those with comorbid diseases or overlap syndromes such as diabetes, liver disease, malignancies, anti-glomerular basement membrane nephritis or immune complex-mediated diseases. A renal biopsy was performed in all subjects before the initial immunosuppressive therapy. All patients received the standard induction treatment with cyclophosphamide and glucocorticoids or rituximab, followed by maintenance therapy with azathioprine and prednisolone [5, 24]. Sample preparation The two-stage sample collection was performed as described below. In the first stage, urine and blood samples were collected [19] in the morning on the day of biopsy in patients with ANCA-GN; patients with proteinuria due to nonvasculitic glomerular diseases [i.e. focal segmental glomerulosclerosis (FSGS, n = 20) and membranous nephropathy (MN, n = 20)] and patients with acute renal failure due to nonglomerular diseases [i.e. acute tubular necrosis (ATN, n = 20)]. Serum and urine samples were also collected from sex- and age-matched healthy volunteers (n = 20) and patients with chronic renal failure due to nonglomerular diseases [i.e. chronic obstructive nephropathy (CON, n = 20)]. None of the patients was being treated with RAS inhibitors at the time of sample collection. In the second stage, serum and urine samples were collected from subjects in the test set every 4 months for up to 24 months (Figure 1). We excluded patients who were being treated with RAS inhibitors during the follow-up. FIGURE 1: View largeDownload slide Flow chart demonstrates the patients enrolled and their follow-up. In the test set, 69 patients were included. All patients were MPO-ANCA positive and had a concomitant renal biopsy performed before initiation of immunosuppressive treatment. Paired urine and blood samples were collected on the day of renal biopsy. A 24-month follow-up was achieved in 50 patients with ANCA-GN. Twenty-eight patients had 4-monthly urine samples taken. In the validation set, 25 patients were included from two other centers in Guangzhou. They were screened following the same inclusion and exclusion criteria as that used in the test set. Subjects included in the validation cohort exhibited characteristics similar to those in the test set. Paired urine and blood samples were collected on the day of renal biopsy. FIGURE 1: View largeDownload slide Flow chart demonstrates the patients enrolled and their follow-up. In the test set, 69 patients were included. All patients were MPO-ANCA positive and had a concomitant renal biopsy performed before initiation of immunosuppressive treatment. Paired urine and blood samples were collected on the day of renal biopsy. A 24-month follow-up was achieved in 50 patients with ANCA-GN. Twenty-eight patients had 4-monthly urine samples taken. In the validation set, 25 patients were included from two other centers in Guangzhou. They were screened following the same inclusion and exclusion criteria as that used in the test set. Subjects included in the validation cohort exhibited characteristics similar to those in the test set. Paired urine and blood samples were collected on the day of renal biopsy. Details of histological and laboratory measurements are described in the Supplementary data. Statistical analyses Statistical analysis was conducted using SPSS 17.0 for Windows (SPSS, Chicago, IL, USA). Continuous variables were expressed as mean ± standard deviation or median (interquartile range). Categorical variables were expressed as percentages. In the case of multiple comparison, one-way analysis of variance (ANOVA) followed by the least square difference test or the Kruskal–Wallis test followed by Dunn’s posttest were performed. Correlations were determined using Pearson’s or Spearman’s correlation coefficient. In these correlation analyses, UAGT was logarithmically transformed to correct for dispersion of data. The association between crescentic ANCA-GN and UAGT was further confirmed using multiple logistic regression analyses by controlling the effect of clinically important confounding variables such as age, and estimated glomerular filtration rate (eGFR) at the time of biopsy. To compare the performance of UAGT and existing clinical markers at different cutoff values, an area under the receiver operating characteristics (ROC) curve (AUC) was generated and C-statistics analysis was performed [25]. Inter- and intraobserver reproducibility of histological analyses were determined by corresponding intraclass correlation coefficients and their 95% confidence intervals (CIs) [26]. P < 0.05 was considered statistically significant. RESULTS Patients with ANCA-GN In the first stage, 69 ANCA-GN patients were enrolled in the test set. All patients were myeloperoxidase (MPO)-ANCA positive and had a concomitant renal biopsy before initiation of immunosuppressive treatment. Paired urine and blood samples were collected on the day of biopsy. Of the patients enrolled, 6 (8.7%) were diagnosed as focal class, 16 (23.2%) as crescentic class, 15 (21.7%) as sclerotic class and 32 (46.4%) as mixed class, according to the histological system proposed by Berden et al. [2]. Clinical features stratified per classification group are presented in Table 1. eGFR at the time of biopsy was found to be higher in the focal class versus all other classes. There was no difference among groups with respect to age, gender, body mass index and hypertension. Table 1. Characteristics of ANCA-GN patients by the renal histological class in the test set Focal Crescentic Mixed Sclerosis P-value (n = 6) (n = 16) (n = 32) (n = 15) Characteristics on admission  Age (years) 36 ± 7 47 ± 11 41 ± 13 47 ± 13 0.106  Men, n (%) 3 (50) 7 (43.7) 15 (46.8) 5 (33) 0.915  Hypertension, n (%) 1 (16.7) 1 (6.25) 4 (12.5) 2 (13.3) 0.881  BMI (kg/m2) 21.5 ± 2.4 21.9 ± 2.9 21.0 ± 2.4 20.5 ± 2.7 0.198 Characteristics at the time of biopsy  SCr (mg/dL) 0.9 ± 0.3 5.0 ± 2.7 4.1 ± 2.7 4.1 ± 2.0 0.001  eGFR (mL/min/1.73 m) 82.6 ± 28.7 16.4 ± 12.1 29.1 ± 22.7 24.1 ± 16.5 0.001  MPO-ANCA (IU/mL) 221.4 ± 97.5 249.9 ± 129.9 277.7 ± 146.5 371.6 ± 169.1 0.272  Ualb (mg/mg Cr) 0.8 ± 0.4 3.4 ± 1.9 2.1 ± 1.0 2.2 ± 1.3 0.001 Focal Crescentic Mixed Sclerosis P-value (n = 6) (n = 16) (n = 32) (n = 15) Characteristics on admission  Age (years) 36 ± 7 47 ± 11 41 ± 13 47 ± 13 0.106  Men, n (%) 3 (50) 7 (43.7) 15 (46.8) 5 (33) 0.915  Hypertension, n (%) 1 (16.7) 1 (6.25) 4 (12.5) 2 (13.3) 0.881  BMI (kg/m2) 21.5 ± 2.4 21.9 ± 2.9 21.0 ± 2.4 20.5 ± 2.7 0.198 Characteristics at the time of biopsy  SCr (mg/dL) 0.9 ± 0.3 5.0 ± 2.7 4.1 ± 2.7 4.1 ± 2.0 0.001  eGFR (mL/min/1.73 m) 82.6 ± 28.7 16.4 ± 12.1 29.1 ± 22.7 24.1 ± 16.5 0.001  MPO-ANCA (IU/mL) 221.4 ± 97.5 249.9 ± 129.9 277.7 ± 146.5 371.6 ± 169.1 0.272  Ualb (mg/mg Cr) 0.8 ± 0.4 3.4 ± 1.9 2.1 ± 1.0 2.2 ± 1.3 0.001 eGFR determined by CKD Epidemiology Collaboration (CKD-EPI) equation. Values presented as mean ± SD unless stated otherwise. CR, creatinine; SCr, serum creatinine. Table 1. Characteristics of ANCA-GN patients by the renal histological class in the test set Focal Crescentic Mixed Sclerosis P-value (n = 6) (n = 16) (n = 32) (n = 15) Characteristics on admission  Age (years) 36 ± 7 47 ± 11 41 ± 13 47 ± 13 0.106  Men, n (%) 3 (50) 7 (43.7) 15 (46.8) 5 (33) 0.915  Hypertension, n (%) 1 (16.7) 1 (6.25) 4 (12.5) 2 (13.3) 0.881  BMI (kg/m2) 21.5 ± 2.4 21.9 ± 2.9 21.0 ± 2.4 20.5 ± 2.7 0.198 Characteristics at the time of biopsy  SCr (mg/dL) 0.9 ± 0.3 5.0 ± 2.7 4.1 ± 2.7 4.1 ± 2.0 0.001  eGFR (mL/min/1.73 m) 82.6 ± 28.7 16.4 ± 12.1 29.1 ± 22.7 24.1 ± 16.5 0.001  MPO-ANCA (IU/mL) 221.4 ± 97.5 249.9 ± 129.9 277.7 ± 146.5 371.6 ± 169.1 0.272  Ualb (mg/mg Cr) 0.8 ± 0.4 3.4 ± 1.9 2.1 ± 1.0 2.2 ± 1.3 0.001 Focal Crescentic Mixed Sclerosis P-value (n = 6) (n = 16) (n = 32) (n = 15) Characteristics on admission  Age (years) 36 ± 7 47 ± 11 41 ± 13 47 ± 13 0.106  Men, n (%) 3 (50) 7 (43.7) 15 (46.8) 5 (33) 0.915  Hypertension, n (%) 1 (16.7) 1 (6.25) 4 (12.5) 2 (13.3) 0.881  BMI (kg/m2) 21.5 ± 2.4 21.9 ± 2.9 21.0 ± 2.4 20.5 ± 2.7 0.198 Characteristics at the time of biopsy  SCr (mg/dL) 0.9 ± 0.3 5.0 ± 2.7 4.1 ± 2.7 4.1 ± 2.0 0.001  eGFR (mL/min/1.73 m) 82.6 ± 28.7 16.4 ± 12.1 29.1 ± 22.7 24.1 ± 16.5 0.001  MPO-ANCA (IU/mL) 221.4 ± 97.5 249.9 ± 129.9 277.7 ± 146.5 371.6 ± 169.1 0.272  Ualb (mg/mg Cr) 0.8 ± 0.4 3.4 ± 1.9 2.1 ± 1.0 2.2 ± 1.3 0.001 eGFR determined by CKD Epidemiology Collaboration (CKD-EPI) equation. Values presented as mean ± SD unless stated otherwise. CR, creatinine; SCr, serum creatinine. Twenty-five ANCA-GN patients were included in the validation set and exhibited characteristics similar to those in the test set (Supplementary data, Table S1). We also included patients with proteinuria due to nonvasculitic glomerular diseases (FSGS and MN) and patients with impaired renal function due to nonglomerular diseases (ATN and CON). Clinical details of these patients are listed in Supplementary data, Table S2. In the second stage, UAGT, 24-h proteinuria and serum creatinine and MPO-ANCA were monitored in 50 subjects with ANCA-GN in the test set for up to 24 months (Figure 1). Urinary AGT level correlated with renal pathological activity in ANCA-GN In the test set, UAGT on the day of biopsy was determined in patients with ANCA-GN, in healthy volunteers and in patients with other renal diseases. Patients with ANCA-GN had significantly greater UAGT [median 355.27 ng/mg of creatinine (first and third quartiles, 158.42 and 614.85)] compared with healthy volunteers [1.39 ng/mg of creatinine (0.92 and 2.78)], patients with FSGS [44.57 ng/mg of creatinine (27.67 and 62.22)], patients with MN [55.86 ng/mg of creatinine (31.86 and 72.29)], patients with ATN [73.63 ng/mg of creatinine (49.44 and 93.36)] and patients with CON [87.56 ng/mg of creatinine (65.05 and 102.56)] (all P < 0.001; Supplementary data, Figure S1A). In contrast to urinalysis, serum AGT levels were comparable among groups (Supplementary data, Figure S1B). There was no correlation between serum and UAGT (r = 0.067, P = 0.387; Supplementary data, Figure S1C). In the test set, UAGT in ANCA-GN on the day of biopsy was compared with the histological parameters for active renal injury (cellular crescents, endocapillary proliferation, fibrinoid necrosis and interstitial inflammation) and parameters for chronic renal injury (glomerular sclerosis, fibrous crescents, tubular atrophy and interstitial fibrosis). As shown in Figure 2, UAGT closely correlated with the extent of active renal injury, particularly the percentages of glomeruli showing cellular crescents (r = 0.616, P < 0.001; Figure 2A) and the severity of acute interstitial inflammation (r = 0.502, P < 0.001; Figure 2B). The extent of chronic renal injury did not correlate with UAGT (Supplementary data, Table S3). FIGURE 2: View largeDownload slide UAGT serves as an indicator of active crescentic ANCA-GN in the test set. (A) Correlation between UAGT level at the time of biopsy and the percentage of glomeruli showing cellular crescents in ANCA-GN. (B) Correlation between UAGT level at the time of biopsy and the extent of acute interstitial inflammation in ANCA-GN. (C and D) Sixty-nine ANCA-GN patients were subgrouped into focal, crescentic, mix and sclerotic classes, following the histological system proposed by Berden. (C) Urinary and (D) serum AGT in patients with different classes of ANCA-GN at the time of biopsy and in healthy volunteers. (E) ROC curve analysis to determine the cutoff value for UAGT and Ualb with respect to diagnosis of crescentic ANCA-GN. (F) ROC curve analysis for the test and validation sets. *P<0.05 versus healthy volunteers in (C) and (D). FIGURE 2: View largeDownload slide UAGT serves as an indicator of active crescentic ANCA-GN in the test set. (A) Correlation between UAGT level at the time of biopsy and the percentage of glomeruli showing cellular crescents in ANCA-GN. (B) Correlation between UAGT level at the time of biopsy and the extent of acute interstitial inflammation in ANCA-GN. (C and D) Sixty-nine ANCA-GN patients were subgrouped into focal, crescentic, mix and sclerotic classes, following the histological system proposed by Berden. (C) Urinary and (D) serum AGT in patients with different classes of ANCA-GN at the time of biopsy and in healthy volunteers. (E) ROC curve analysis to determine the cutoff value for UAGT and Ualb with respect to diagnosis of crescentic ANCA-GN. (F) ROC curve analysis for the test and validation sets. *P<0.05 versus healthy volunteers in (C) and (D). UAGT as an indicator of crescentic ANCA-GN In the test set, UAGT was compared among different histological groups. In crescentic ANCA-GN, UAGT dramatically increased [1120.51 ng/mg of creatinine (first and third quartiles, 483.33 and 1828.08)] compared with mixed [325.46 ng/mg of creatinine (200.26 and 459.06)], sclerotic [202.35 ng/mg of creatinine (90.25 and 406.35)] or focal ANCA-GN [115.26 ng/mg of creatinine (60.71 and 272.58)] (all P < 0.001; Figure 2C). Serum AGT levels were comparable among different histological groups (Figure 2D). UAGT associated with the existence of crescentic ANCA-GN (Supplementary data, Figure S2). This association was supported by the multivariable analyses. After adjustment for clinical variables including age and eGFR on the day of biopsy, UAGT was the most powerful indicator of crescentic ANCA-GN. The highest quartile of UAGT on the day of biopsy was associated with an increased risk for crescentic ANCA-GN by 6-fold compared with the lowest quartile (Supplementary data, Table S4 and Figure S2). To assess the diagnostic value of UAGT for crescentic ANCA-GN, an ROC curve was generated. The AUC of UAGT on the day of biopsy for diagnosing crescentic ANCA-GN was 0.88, which was greater than those of existing clinical markers, such as urinary albumin:creatinine ratio (Ualb, 0.73, P = 0.029), eGFR (0.69, P = 0.009), MPO-ANCA (0.42, P < 0.001) and the combined clinical markers (0.79, P = 0.039) (Figure 2E and Supplementary data, Table S5). We further evaluated the performance of a combination of UAGT and the clinical markers in the test set. When UAGT was added to the clinical markers, the performance for identifying crescentic ANCA-GN was further improved (all P < 0.05) (Table 2). Table 2. Performance of a combination of UAGT and clinical markers for diagnosing crescentic ANCA-GN in the test set Clinical marker AUC (95%) Clinical marker Clinical marker and UAGT P-value Ualb 0.729 (0.591–0.868) 0.893 (0.789–0.996) 0.009 eGFR 0.690 (0.560–0.820) 0.890 (0.785–0.996) 0.001 MPO-ANCA 0.422 (0.255–0.590) 0.874 (0.755–0.993) 0.010 Combination of clinical markers 0.795 (0.681–0.883) 0.894 (0.796–0.955) 0.034 Clinical marker AUC (95%) Clinical marker Clinical marker and UAGT P-value Ualb 0.729 (0.591–0.868) 0.893 (0.789–0.996) 0.009 eGFR 0.690 (0.560–0.820) 0.890 (0.785–0.996) 0.001 MPO-ANCA 0.422 (0.255–0.590) 0.874 (0.755–0.993) 0.010 Combination of clinical markers 0.795 (0.681–0.883) 0.894 (0.796–0.955) 0.034 eGFR determined by CKD Epidemiology Collaboration (CKD-EPI) equation. Table 2. Performance of a combination of UAGT and clinical markers for diagnosing crescentic ANCA-GN in the test set Clinical marker AUC (95%) Clinical marker Clinical marker and UAGT P-value Ualb 0.729 (0.591–0.868) 0.893 (0.789–0.996) 0.009 eGFR 0.690 (0.560–0.820) 0.890 (0.785–0.996) 0.001 MPO-ANCA 0.422 (0.255–0.590) 0.874 (0.755–0.993) 0.010 Combination of clinical markers 0.795 (0.681–0.883) 0.894 (0.796–0.955) 0.034 Clinical marker AUC (95%) Clinical marker Clinical marker and UAGT P-value Ualb 0.729 (0.591–0.868) 0.893 (0.789–0.996) 0.009 eGFR 0.690 (0.560–0.820) 0.890 (0.785–0.996) 0.001 MPO-ANCA 0.422 (0.255–0.590) 0.874 (0.755–0.993) 0.010 Combination of clinical markers 0.795 (0.681–0.883) 0.894 (0.796–0.955) 0.034 eGFR determined by CKD Epidemiology Collaboration (CKD-EPI) equation. To validate the diagnostic value of UAGT for crescentic ANCA-GN, the AUC for identifying crescentic ANCA-GN was analyzed in an independent validation set. As demonstrated in Figure 2F, the ability of UAGT to diagnose crescentic ANCA-GN was validated in the validation set (AUC = 0.81). UAGT as a marker for treatment response and renal relapse Of the 69 ANCA-GN patients in the test set, 19 were excluded (9 died, 8 progressed to end-stage renal disease and 2 were lost to follow-up). Therefore, in the second stage, renal function and UAGT were monitored in 50 ANCA-GN patients for up to 24 months (Figure 1). All the patients received the standard induction treatment with cyclophosphamide and glucocorticoids or rituximab, followed by maintenance therapy with azathioprine and prednisolone. Renal relapse is defined as an initiation of immunosuppressive rescue therapy due to an increasing serum creatinine and an increasing number of urinary red cells [27]. As shown in Figure 3A, UAGT significantly decreased during immunosuppressive treatment and remained low during follow-up (P < 0.001). The reduction in UAGT was even more pronounced when patients with relapsing disease were excluded during follow-up (Figure 3B). FIGURE 3: View largeDownload slide UAGT levels in ANCA-GN during follow-up in the test set. (A) Urinary levels of AGT decreased during the initial immunosuppressive treatment and remained low during the maintenance therapy. The AGT measurements obtained at the time of biopsy (n = 69) and after 8 months (n = 64), 16 months (n = 46) and 24 months (n = 50) of follow-up. (B) UAGT measured during the 24-month follow-up excluding patients with relapsing disease (n = 62 at the time of biopsy, n = 57 after 8 months, n = 39 after 16 months and n = 43 after 24 months). (C–E) Longitudinal study with measurements of UAGT in patients suffering from renal relapse. Arrows indicate the onset of relapse. *P < 0.05 in (A) and (B). FIGURE 3: View largeDownload slide UAGT levels in ANCA-GN during follow-up in the test set. (A) Urinary levels of AGT decreased during the initial immunosuppressive treatment and remained low during the maintenance therapy. The AGT measurements obtained at the time of biopsy (n = 69) and after 8 months (n = 64), 16 months (n = 46) and 24 months (n = 50) of follow-up. (B) UAGT measured during the 24-month follow-up excluding patients with relapsing disease (n = 62 at the time of biopsy, n = 57 after 8 months, n = 39 after 16 months and n = 43 after 24 months). (C–E) Longitudinal study with measurements of UAGT in patients suffering from renal relapse. Arrows indicate the onset of relapse. *P < 0.05 in (A) and (B). Among 50 patients who were followed up for 24 months, 7 of them suffered from relapsing ANCA-GN. In three of the seven patients, UAGT was assessed at the onset of relapse. The renal relapse was associated with an elevation in UAGT (Figure 3C–E). The corresponding UAGT, 24-h proteinuria, serum creatinine and MPO-ANCA are presented in Supplementary data, Table S6. UAGT reflects intrarenal RAS status To investigate the relationship between UAGT and renal production of AGT, immunohistochemistry of AGT and angiotensin II was performed in renal biopsies from ANCA-GN patients. Immunoreactivity of AGT and angiotensin II significantly increased in biopsies of crescentic ANCA-GN compared with that in normal kidneys, and in biopsies of mixed, sclerotic or focal ANCA-GN (all P < 0.001; Figure 4). Overexpression of AGT in biopsies was observed predominantly in proximal tubules and cellular crescents (Figure 4A). Staining of angiotensin II was detected mainly in distal tubules and cellular crescents (Figure 4A). Glomerular and tubular expression of AGT or angiotensin II positively correlated with UAGT on the day of biopsy (Figure 4C, E). FIGURE 4: View largeDownload slide Expression of intrarenal RAS correlates with UAGT level in patients with ANCA-GN. (A) Representative photos of intrarenal AGT and angiotensin II (Ang II) expression in glomeruli and tubulointerstitium shown by immunohistochemical staining. (B) Semiquantitative data of intrarenal AGT staining. (C) Correlation between glomerular or tubulointerstitial AGT expression and UAGT levels. (D) Semiquantitative data of intrarenal Ang II staining. (E) Correlation between glomerular or tubulointerstitial Ang II expression and UAGT levels. Scale bar = 100 µm. *P < 0.05 versus normal kidney in (B) and (D). FIGURE 4: View largeDownload slide Expression of intrarenal RAS correlates with UAGT level in patients with ANCA-GN. (A) Representative photos of intrarenal AGT and angiotensin II (Ang II) expression in glomeruli and tubulointerstitium shown by immunohistochemical staining. (B) Semiquantitative data of intrarenal AGT staining. (C) Correlation between glomerular or tubulointerstitial AGT expression and UAGT levels. (D) Semiquantitative data of intrarenal Ang II staining. (E) Correlation between glomerular or tubulointerstitial Ang II expression and UAGT levels. Scale bar = 100 µm. *P < 0.05 versus normal kidney in (B) and (D). To localize AGT-expressing cells in the kidney, double staining was performed in biopsies of patients with crescentic ANCA-GN. Overexpression of AGT was localized mainly in proximal tubular cells, infiltrated macrophages, podocytes and parietal epithelial cells (Figure 5). FIGURE 5: View largeDownload slide Overexpression of AGT in crescentic ANCA-GN was localized mainly in proximal tubular cells, infiltrated macrophages, podocytes and parietal epithelial cells. Representative photographs of AGT localization determined with double staining of antibody against AGT and antibodies against markers of renal tubular segments, markers of macrophages, markers of podocytes or markers of parietal epithelial cells. Scale bar = 100 µm. AQP1, aquaporin 1 (proximal tubule); THP, Tamm–Horsfall protein (thick ascending limb); AQP2, NCCT or thiazide-sensitive NaCl cotransporter (distal tubule); aquaporin 2 (collecting duct); CD68 (macrophage); WT1, Wilms’ tumor (podocyte); Claudin 1 (parietal epithelial cell). FIGURE 5: View largeDownload slide Overexpression of AGT in crescentic ANCA-GN was localized mainly in proximal tubular cells, infiltrated macrophages, podocytes and parietal epithelial cells. Representative photographs of AGT localization determined with double staining of antibody against AGT and antibodies against markers of renal tubular segments, markers of macrophages, markers of podocytes or markers of parietal epithelial cells. Scale bar = 100 µm. AQP1, aquaporin 1 (proximal tubule); THP, Tamm–Horsfall protein (thick ascending limb); AQP2, NCCT or thiazide-sensitive NaCl cotransporter (distal tubule); aquaporin 2 (collecting duct); CD68 (macrophage); WT1, Wilms’ tumor (podocyte); Claudin 1 (parietal epithelial cell). DISCUSSION One of the major challenges in optimizing management of ANCA-GN is the lack of a high-performance indicator to ascertain histological lesions and disease activity. Currently assessment of renal injury can be only achieved in kidney biopsies, which is obtained through an invasive procedure [2, 8, 28]. Thus it is difficult to dynamically monitor renal disease activity, which may allow for evaluation of therapy efficacy and renal relapse in clinical circumstances. In the present study, we demonstrate that UAGT paralleled kidney RAS status and renal pathological activity in ANCA-GN. Tracking UAGT could be used to monitor treatment response and renal relapse over time. In the first phase of the study, we found that UAGT, measured on the day of the biopsy, varied significantly depending on the renal pathological activity in ANCA-GN. Recent studies have demonstrated that UAGT reflects the severity of renal histology [18, 19]. However, association between UAGT and renal pathological activity in ANCA-GN has not been established. Our data showed that UAGT at the time of biopsy strongly correlated with the number of cellular crescents and the degree of interstitial inflammation. Such an association between UAGT and active renal damage suggested a potential role of UAGT in identifying active ANCA-GN. This usefulness of UAGT would not be affected by renal function impairment, proteinuria or hypertension. Patients with impaired renal function (i.e. ATN/CON) or sustained proteinuria (i.e. FSGS/MN) exhibited much lower UAGT than those with ANCA-GN. In patients with ANCA-GN, UAGT did not correlated with systolic (r = 0.12, P = 0.318) or diastolic blood pressure (r = 0.13, P = 0.269). More importantly, we demonstrated that UAGT is a novel biomarker for crescentic ANCA-GN. Based on the histological classification schema proposed by Berden et al. [2], ANCA-GN has been classified into four distinct categories: focal, crescentic, mixed and sclerotic. Crescentic ANCA-GN is a particularly aggressive type of ANCA-GN that associated with highly active renal lesions, rapidly declining renal function and a good chance of renal function recovery [2, 10, 29–31]. UAGT was markedly elevated in patients with crescentic ANCA-GN. The performance of UAGT for identifying crescentic ANCN-GN is superior to other existing clinical screening markers such as Ualb, eGFR, MPO-ANCA and the combined clinical markers. Furthermore, we demonstrated that the addition of UAGT to existing clinical markers significantly increased the accuracy of diagnosing crescentic ANCA-GN, as demonstrated by AUC results. The value of UAGT for identifying crescentic ANCA-GN was further demonstrated in a validation cohort. These results support the use of UAGT, especially the combination use of UAGT with other clinical markers, in identifying crescentic ANCA-GN. A large number of clinical studies demonstrate that the presence of specific histological lesions in renal biopsies is helpful in predicting renal outcome and in planning therapy [2, 31–33]. Active glomerular lesions, in particular cellular crescents formation, have been found to correlate with recovery of renal function irrespective of baseline GFR [2, 31]. Therefore, measurement of UAGT is of great help in identifying patients with active renal damage, in particular crescentic ANCA-GN. Timely recognition of active renal involvement would prompt consideration of immunosuppressive treatment to prevent irreversible kidney damage. Monitoring of renal disease activity could be beneficial in clinical practice. Identifying patients with relapsing and persistent disease would help physicians to optimize management and minimize treatment-related side effects [5, 24]. In the second phase of the study, we evaluated the value of UAGT in the monitoring of renal disease activity in ANCA-GN. UAGT generally decreased after the induction of immunotherapy and stayed low during 24 months of follow-up. Patients experiencing renal relapses exhibited an elevation in UAGT. Therefore it is conceivable to speculate that UAGT reflects disease activity and can serve as an indicator of renal relapse in ANCA-GN. Supporting our findings, a previous report demonstrated a reduction in UAGT following steroids therapy in patients with chronic GN [17]. However, whether UAGT is predictive of relapse remains unclear and needs further studies. Both urinary and serum AGT were detected simultaneously in this study. Contrary to UAGT, serum AGT was not elevated in ANCA-GN. Thus UAGT may not reflect systemic RAS status. A potential site of AGT secretion into the urine is the kidney [19]. In the present study, immunohistochemistry analysis revealed a significantly increased expression of AGT and angiotensin II in renal biopsies from patients with crescentic ANCA-GN. UAGT correlated well with intrarenal expression of AGT and angiotensin II, suggesting that increased UAGT excretion in ANCA-GN may be due to enhanced synthesis in the kidney. Consistently, staining of AGT, an important substrate of angiotensin II, has been identified in tubular cells, macrophages, parietal epithelial cells and podocytes, which makes its secretion to the urine possible. Increased production of intrarenal AGT under pathological conditions can be mediated through positive feedback from angiotensin II [34]. As such, UAGT might also be proposed as a marker of intrarenal RAS status. This extends similar findings in patients with chronic kidney disease [11, 17], type 2 diabetes [35], hypertension [36], polycystic disease [37] as well as podocyte injury [38]. Intrarenal RAS has been identified as a key pathogenic mediator of cell proliferation and migration that results in renal inflammation and crescent formation [11–13]. In our study, we demonstrated significantly increased expression of intrarenal RAS that positively correlated with UAGT in crescentic ANCA-GN. This result provides a rationale to use angiotensin receptor blockers to reduce active renal tissue injury in patients with ANCA-GN. Indeed, RAS inhibitor has been demonstrated to improve renal function and induce regression of crescentic lesions in patients with crescentic ANCA-GN [12]. Our present study has the following strengths. First, all subjects enrolled had a concomitant renal biopsy performed before initiation of immunosuppressive treatment. Paired urine and blood samples were collected on the day of biopsy, allowing us to evaluate the correlation between UAGT and renal pathological activity or intrarenal RAS status. Double immunofluorescent staining identified proximal tubules, macrophages, podocytes and parietal epithelium expressing AGT in the kidney. Additionally, we compared the diagnostic performance of UAGT with existing clinical makers in crescentic ANCA-GN. We demonstrated that UAGT is a powerful indicator of crescentic ANCA-GN and outperforms the existing clinical markers. This result has been confirmed in a validation cohort. Finally, we followed the subjects with ANCA-GN every 4 months for 24 months after biopsy, which allowed us to assess the ability of UAGT in indicating disease activity and renal relapse in ANCA-GN. This study also has some limitations. First, only patients not being treated with RAS inhibitors were enrolled, therefore the influence of RAS blockade on the value of UAGT as a biomarker of disease activity remains to be investigated in our ongoing serial studies. Second, we did not include patients with active nonrenal ANCA-associated vasculitis and thus cannot exclude the contribution of AGT from the circulation into the urine. However, serum AGT was not elevated in ANCA-GN patients and did not correlated with UAGT. Therefore it seems unlikely that the increased UAGT in ANCA-GN was mainly due to filtered AGT from the systemic circulation. In addition, urinary, instead of serum AGT, serves as an indicator of renal disease activity. In clinical settings there are circumstances, however, in which urine samples are unavailable (i.e. patients with oliguria). Moreover, all ANCA-GN patients included were MPO-ANCA positive. Since there is a striking predominance of MPO-ANCA over proteinase 3 (PR3)-ANCA in Chinese people [39], validation of our findings in cohorts with enough patients with PR3-ANCA-GN would be desirable. In conclusion, our study shows that UAGT could serve as a useful biomarker for renal pathology activity and could be used to monitor treatment response and renal relapse. Application of this novel biomarker clinically would allow for evaluation of therapy efficacy and minimization of treatment-related toxicity in ANCA-GN. SUPPLEMENTARY DATA Supplementary data are available at ndt online. FUNDING This work was supported by the National Natural Science Foundation of China (81570619). AUTHORS’ CONTRIBUTIONS L.W. performed the experiments and evaluated and interpreted data. M.Y., L.J. and X.F. performed histological and biochemical experiments, evaluated data and contributed to manuscript preparation. Z.Z., M.Y., X.Z., M.S., S.C., C.W. and Z.Y. performed biochemical experiments and contributed to manuscript preparation. S.C. and C.W. helped to enroll patients in the validation set. W.C. designed, supervised and financed the study and wrote the manuscript. All authors have reviewed and revised the manuscript. CONFLICT OF INTEREST STATEMENT None declared. REFERENCES 1 Jennette JC , Falk RJ. Small-vessel vasculitis . N Engl J Med 1997 ; 337 : 1512 – 1523 Google Scholar CrossRef Search ADS PubMed 2 Berden AE , Ferrario F , Hagen EC et al. Histopathologic classification of ANCA-associated glomerulonephritis . J Am Soc Nephrol 2010 ; 21 : 1628 – 1636 Google Scholar CrossRef Search ADS PubMed 3 Jones RB , Tervaert JW , Hauser T et al. Rituximab versus cyclophosphamide in ANCA-associated renal vasculitis . N Engl J Med 2010 ; 363 : 211 – 220 Google Scholar CrossRef Search ADS PubMed 4 Specks U , Merkel PA , Seo P et al. Efficacy of remission-induction regimens for ANCA-associated vasculitis . N Engl J Med 2013 ; 369 : 417 – 427 Google Scholar CrossRef Search ADS PubMed 5 Schonermarck U , Gross WL , de Groot K. Treatment of ANCA-associated vasculitis . Nat Rev Nephrol 2014 ; 10 : 25 – 36 Google Scholar CrossRef Search ADS PubMed 6 Little MA , Nightingale P , Verburgh CA et al. Early mortality in systemic vasculitis: relative contribution of adverse events and active vasculitis . Ann Rheum Dis 2010 ; 69 : 1036 – 1043 Google Scholar CrossRef Search ADS PubMed 7 Falk RJ , Jennette JC. ANCA small-vessel vasculitis . J Am Soc Nephrol 1997 ; 8 : 314 – 322 Google Scholar PubMed 8 Rahmattulla C , Bruijn JA , Bajema IM. Histopathological classification of antineutrophil cytoplasmic antibody-associated glomerulonephritis. An update . Curr Opin Nephrol Hypertens 2014 ; 23 : 224 – 231 Google Scholar CrossRef Search ADS PubMed 9 Jennette JC , Falk RJ. Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease . Nat Rev Rheumatol 2014 ; 10 : 463 – 473 Google Scholar CrossRef Search ADS PubMed 10 Singh SK , Jeansson M , Quaggin SE. New insights into the pathogenesis of cellular crescents . Curr Opin Nephrol Hypertens 2011 ; 20 : 258 – 262 Google Scholar CrossRef Search ADS PubMed 11 Kobori H , Nangaku M , Navar LG et al. The intrarenal renin–angiotensin system: from physiology to the pathobiology of hypertension and kidney disease . Pharmacol Rev 2007 ; 59 : 251 – 287 Google Scholar CrossRef Search ADS PubMed 12 Rizzo P , Perico N , Gagliardini E et al. Nature and mediators of parietal epithelial cell activation in glomerulonephritides of human and rat . Am J Pathol 2013 ; 183 : 1769 – 1778 Google Scholar CrossRef Search ADS PubMed 13 Aki K , Shimizu A , Masuda Y et al. ANG II receptor blockade enhances anti-inflammatory macrophages in anti-glomerular basement membrane glomerulonephritis . Am J Physiol Renal Physiol 2010 ; 298 : F870 – F882 Google Scholar CrossRef Search ADS PubMed 14 Urushihara M , Ohashi N , Miyata K et al. Addition of angiotensin II type 1 receptor blocker to CCR2 antagonist markedly attenuates crescentic glomerulonephritis . Hypertension 2011 ; 57 : 586 – 593 Google Scholar CrossRef Search ADS PubMed 15 Kinoshita Y , Kondo S , Urushihara M et al. Angiotensin II type I receptor blockade suppresses glomerular renin–angiotensin system activation, oxidative stress, and progressive glomerular injury in rat anti-glomerular basement membrane glomerulonephritis . Transl Res 2011 ; 158 : 235 – 248 Google Scholar CrossRef Search ADS PubMed 16 Minutolo R , Balletta MM , Catapano F et al. Mesangial hypercellularity predicts antiproteinuric response to dual blockade of RAS in primary glomerulonephritis . Kidney Int 2006 ; 70 : 1170 – 1176 Google Scholar CrossRef Search ADS PubMed 17 Urushihara M , Kondo S , Kagami S et al. Urinary angiotensinogen accurately reflects intrarenal renin–angiotensin system activity . Am J Nephrol 2010 ; 31 : 318 – 325 Google Scholar CrossRef Search ADS PubMed 18 Kim SM , Jang HR , Lee YJ et al. Urinary angiotensinogen levels reflect the severity of renal histopathology in patients with chronic kidney disease . Clin Nephrol 2011 ; 76 : 117 – 123 Google Scholar CrossRef Search ADS PubMed 19 Yamamoto T , Nakagawa T , Suzuki H et al. Urinary angiotensinogen as a marker of intrarenal angiotensin II activity associated with deterioration of renal function in patients with chronic kidney disease . J Am Soc Nephrol 2007 ; 18 : 1558 – 1565 Google Scholar CrossRef Search ADS PubMed 20 Cao W , Jin L , Zhou Z et al. Overexpression of intrarenal renin–angiotensin system in human acute tubular necrosis . Kidney Blood Press Res 2016 ; 41 : 746 – 756 Google Scholar CrossRef Search ADS PubMed 21 Kobori H , Harrison-Bernard LM , Navar LG. Urinary excretion of angiotensinogen reflects intrarenal angiotensinogen production . Kidney Int 2002 ; 61 : 579 – 585 Google Scholar CrossRef Search ADS PubMed 22 Kobori H , Katsurada A , Ozawa Y et al. Enhanced intrarenal oxidative stress and angiotensinogen in IgA nephropathy patients . Biochem Biophys Res Commun 2007 ; 358 : 156 – 163 Google Scholar CrossRef Search ADS PubMed 23 Kobori H , Ohashi N , Katsurada A et al. Urinary angiotensinogen as a potential biomarker of severity of chronic kidney diseases . J Am Soc Hypertens 2008 ; 2 : 349 – 354 Google Scholar CrossRef Search ADS PubMed 24 Moroni G , Ponticelli C. Rapidly progressive crescentic glomerulonephritis: early treatment is a must . Autoimmun Rev 2014 ; 13 : 723 – 729 Google Scholar CrossRef Search ADS PubMed 25 DeLong ER , DeLong DM , Clarke-Pearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach . Biometrics 1988 ; 44 : 837 – 845 Google Scholar CrossRef Search ADS PubMed 26 Shrout PE , Fleiss JL. Intraclass correlations: uses in assessing rater reliability . Psychol Bull 1979 ; 86 : 420 – 428 Google Scholar CrossRef Search ADS PubMed 27 Brix SR , Stege G , Disteldorf E et al. CC chemokine ligand 18 in ANCA-associated crescentic GN . J Am Soc Nephrol 2015 ; 26 : 2105 – 2117 Google Scholar CrossRef Search ADS PubMed 28 de Lind van Wijngaarden RA , Hauer HA , Wolterbeek R et al. Chances of renal recovery for dialysis-dependent ANCA-associated glomerulonephritis . J Am Soc Nephrol 2007 ; 18 : 2189 – 2197 Google Scholar CrossRef Search ADS PubMed 29 Iwakiri T , Fujimoto S , Kitagawa K et al. Validation of a newly proposed histopathological classification in Japanese patients with anti-neutrophil cytoplasmic antibody-associated glomerulonephritis . BMC Nephrol 2013 ; 14 : 125 Google Scholar CrossRef Search ADS PubMed 30 Lionaki S , Mavragani CP , Karras A et al. Predictors of renal histopathology in antineutrophil cytoplasmic antibody associated glomerulonephritis . J Autoimmun 2016 ; 72 : 57 – 64 Google Scholar CrossRef Search ADS PubMed 31 Hauer HA , Bajema IM , Van Houwelingen HC et al. Determinants of outcome in ANCA-associated glomerulonephritis: a prospective clinico-histopathological analysis of 96 patients . Kidney Int 2002 ; 62 : 1732 – 1742 Google Scholar CrossRef Search ADS PubMed 32 de Lind van Wijngaarden RA , Hauer HA , Wolterbeek R et al. Clinical and histologic determinants of renal outcome in ANCA-associated vasculitis: a prospective analysis of 100 patients with severe renal involvement . J Am Soc Nephrol 2006 ; 17 : 2264 – 2274 Google Scholar CrossRef Search ADS PubMed 33 Neumann I , Kain R , Regele H et al. Histological and clinical predictors of early and late renal outcome in ANCA-associated vasculitis . Nephrol Dial Transplant 2005 ; 20 : 96 – 104 Google Scholar CrossRef Search ADS PubMed 34 Kobori H , Ozawa Y , Suzaki Y et al. Young scholars award lecture: intratubular angiotensinogen in hypertension and kidney diseases . Am J Hypertens 2006 ; 19 : 541 – 550 Google Scholar CrossRef Search ADS PubMed 35 Persson F , Lu X , Rossing P et al. Urinary renin and angiotensinogen in type 2 diabetes: added value beyond urinary albumin? J Hypertens 2013 ; 31 : 1646 – 1652 Google Scholar CrossRef Search ADS PubMed 36 Michel FS , Norton GR , Maseko MJ et al. Urinary angiotensinogen excretion is associated with blood pressure independent of the circulating renin–angiotensin system in a group of African ancestry . Hypertension 2014 ; 64 : 149 – 156 Google Scholar CrossRef Search ADS PubMed 37 Salih M , Bovee DM , Roksnoer LCW et al. Urinary renin–angiotensin markers in polycystic kidney disease . Am J Physiol Renal Physiol 2017 ; 313 : F874 – F881 Google Scholar CrossRef Search ADS PubMed 38 Eriguchi M , Yotsueda R , Torisu K et al. Assessment of urinary angiotensinogen as a marker of podocyte injury in proteinuric nephropathies . Am J Physiol Renal Physiol 2016 ; 310 : F322 – F333 Google Scholar CrossRef Search ADS PubMed 39 Liu LJ , Chen M , Yu F et al. Evaluation of a new algorithm in classification of systemic vasculitis . Rheumatology (Oxford) 2008 ; 47 : 708 – 712 Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nephrology Dialysis Transplantation Oxford University Press

Urinary angiotensinogen: an indicator of active antineutrophil cytoplasmic antibody-associated glomerulonephritis

Loading next page...
 
/lp/ou_press/urinary-angiotensinogen-an-indicator-of-active-antineutrophil-tO1yYtcZ0E
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
ISSN
0931-0509
eISSN
1460-2385
D.O.I.
10.1093/ndt/gfy112
Publisher site
See Article on Publisher Site

Abstract

Abstract Background One of the major challenges in improving the management of antineutrophil cytoplasmic antibody–associated glomerulonephritis (ANCA-GN) is the lack of a disease-specific indicator for histological lesions and disease activity. Here we tested the utility of urinary angiotensinogen (UAGT) as a biomarker of renal disease activity in ANCA-GN. Methods A prospective, two-stage cohort study was performed in ANCA-GN patients. In Stage I, UAGT was measured at the time of renal biopsy in 69 patients from two centers (test set) and 25 patients from two other centers (validation set). In Stage II, UAGT was monitored in 50 subjects in the test set for 24 months. Results In Stage I, UAGT significantly increased in ANCA-GN patients, correlating well with cellular crescents formation and active interstitial inflammation. Patients with crescentic ANCA-GN exhibited the highest UAGT compared with other histopathological classes of ANCA-GN. After multivariable adjustment, the highest quartile of UAGT, compared with the lowest quartile, associated with a 6-fold increased risk of crescentic ANCA-GN. For predicting crescentic ANCA-GN, UAGT [area under the receiver operating characteristics curve (AUC) = 0.88] outperformed albuminuria (AUC = 0.73) and estimated glomerular filtration rate (AUC = 0.69). UAGT improved the performance of those clinical markers in diagnosing crescentic ANCA-GN (P < 0.034), suggesting a role of UAGT in identifying active crescentic ANCA-GN. In Stage II, UAGT decreased after immunotherapy and increased at the time of renal relapse during the 2-year follow-up, suggesting the usefulness of UAGT to monitor disease activity over time. Conclusions These results suggest the potential use of UAGT for assessing disease activity and renal relapse in ANCA-GN. ANCA glomerulonephritis, disease activity, indicator, renal pathology, urinary angiotensinogen INTRODUCTION Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is the leading cause of rapidly progressive crescentic glomerulonephritis (GN) [1, 2]. Current therapeutic strategies have substantial side effects and relapses are frequent [3–5]. The mortality of patients in the first year of therapy is from adverse events rather than from the disease itself [6]. Investigations that can be used to detect renal disease activity and treatment response are helpful in optimizing therapy and minimizing treatment-associated side effects. The widespread use of ANCA assays facilitates the diagnosis of ANCA-GN, but the ANCA level does not correlate well with renal disease activity [7]. Assessments of renal disease activity are only achievable with renal biopsies, which are obtained from patients via an invasive operation [2, 8]. In this setting, development of a noninvasive surrogate biomarker that closely parallels renal pathological activity is urgently needed. The pathogenesis of ANCA-GN is multifactorial but is likely related to necrotizing vascular inflammation as well as crescents formation in glomeruli [9, 10]. There is substantial evidence that activation of the intrarenal renin–angiotensin system (RAS) drives leukocyte recruitment and renal cell proliferation and thereby plays an important role in the development of proliferative and crescentic GN [11–13]. Indeed, enhanced intrarenal production of angiotensin II and angiotensinogen (AGT) has been observed in animals with crescentic GN [12, 14, 15]. Treatment with RAS blockers markedly ameliorates crescentic lesions in GN [12, 16, 17]. Furthermore, AGT, an important substrate of angiotensin II, can be secreted from AGT-positive cells in the kidney and be detectable in the urine [11]. Urinary AGT (UAGT) has been proposed as a biomarker of renal disease severity [18, 19] and has been shown to associate with renal outcome in certain diseases [19–23]. However, the exact levels of UAGT and their relevance to renal pathology and disease activity in human ANCA-GN remain to be elucidated. We conducted a prospective observational study in 69 patients with biopsy-proven ANCA-GN. Our objective is to test and validate the hypothesis that UAGT could serve as a useful biomarker for renal pathology activity and could be used to monitor treatment response and renal relapse. This is the first study in ANCA-GN to demonstrate the utility of UAGT as an indicator of disease activity and renal relapse. MATERIALS AND METHODS Patients This study was performed in accordance with the Declaration of Helsinki and was approved by the institutional ethical review board for each center. All the participants provided written informed consent. Sample collection for the test set was conducted in two centers in Wenzhou and Guangzhou from September 2012 to August 2017. The validation test was conducted in two other centers in Guangzhou from January 2016 to March 2018. Patients with biopsy-proven ANCA-GN in the two sets were enrolled according to the inclusion and exclusion criteria described below. Eligible participants were patients with biopsy-proven ANCA-GN ages 18–80 years. Exclusion criteria include those with inadequate biopsy specimens (<10 glomeruli), those being treated with RAS inhibitors at the time of sample collection, those with comorbid diseases or overlap syndromes such as diabetes, liver disease, malignancies, anti-glomerular basement membrane nephritis or immune complex-mediated diseases. A renal biopsy was performed in all subjects before the initial immunosuppressive therapy. All patients received the standard induction treatment with cyclophosphamide and glucocorticoids or rituximab, followed by maintenance therapy with azathioprine and prednisolone [5, 24]. Sample preparation The two-stage sample collection was performed as described below. In the first stage, urine and blood samples were collected [19] in the morning on the day of biopsy in patients with ANCA-GN; patients with proteinuria due to nonvasculitic glomerular diseases [i.e. focal segmental glomerulosclerosis (FSGS, n = 20) and membranous nephropathy (MN, n = 20)] and patients with acute renal failure due to nonglomerular diseases [i.e. acute tubular necrosis (ATN, n = 20)]. Serum and urine samples were also collected from sex- and age-matched healthy volunteers (n = 20) and patients with chronic renal failure due to nonglomerular diseases [i.e. chronic obstructive nephropathy (CON, n = 20)]. None of the patients was being treated with RAS inhibitors at the time of sample collection. In the second stage, serum and urine samples were collected from subjects in the test set every 4 months for up to 24 months (Figure 1). We excluded patients who were being treated with RAS inhibitors during the follow-up. FIGURE 1: View largeDownload slide Flow chart demonstrates the patients enrolled and their follow-up. In the test set, 69 patients were included. All patients were MPO-ANCA positive and had a concomitant renal biopsy performed before initiation of immunosuppressive treatment. Paired urine and blood samples were collected on the day of renal biopsy. A 24-month follow-up was achieved in 50 patients with ANCA-GN. Twenty-eight patients had 4-monthly urine samples taken. In the validation set, 25 patients were included from two other centers in Guangzhou. They were screened following the same inclusion and exclusion criteria as that used in the test set. Subjects included in the validation cohort exhibited characteristics similar to those in the test set. Paired urine and blood samples were collected on the day of renal biopsy. FIGURE 1: View largeDownload slide Flow chart demonstrates the patients enrolled and their follow-up. In the test set, 69 patients were included. All patients were MPO-ANCA positive and had a concomitant renal biopsy performed before initiation of immunosuppressive treatment. Paired urine and blood samples were collected on the day of renal biopsy. A 24-month follow-up was achieved in 50 patients with ANCA-GN. Twenty-eight patients had 4-monthly urine samples taken. In the validation set, 25 patients were included from two other centers in Guangzhou. They were screened following the same inclusion and exclusion criteria as that used in the test set. Subjects included in the validation cohort exhibited characteristics similar to those in the test set. Paired urine and blood samples were collected on the day of renal biopsy. Details of histological and laboratory measurements are described in the Supplementary data. Statistical analyses Statistical analysis was conducted using SPSS 17.0 for Windows (SPSS, Chicago, IL, USA). Continuous variables were expressed as mean ± standard deviation or median (interquartile range). Categorical variables were expressed as percentages. In the case of multiple comparison, one-way analysis of variance (ANOVA) followed by the least square difference test or the Kruskal–Wallis test followed by Dunn’s posttest were performed. Correlations were determined using Pearson’s or Spearman’s correlation coefficient. In these correlation analyses, UAGT was logarithmically transformed to correct for dispersion of data. The association between crescentic ANCA-GN and UAGT was further confirmed using multiple logistic regression analyses by controlling the effect of clinically important confounding variables such as age, and estimated glomerular filtration rate (eGFR) at the time of biopsy. To compare the performance of UAGT and existing clinical markers at different cutoff values, an area under the receiver operating characteristics (ROC) curve (AUC) was generated and C-statistics analysis was performed [25]. Inter- and intraobserver reproducibility of histological analyses were determined by corresponding intraclass correlation coefficients and their 95% confidence intervals (CIs) [26]. P < 0.05 was considered statistically significant. RESULTS Patients with ANCA-GN In the first stage, 69 ANCA-GN patients were enrolled in the test set. All patients were myeloperoxidase (MPO)-ANCA positive and had a concomitant renal biopsy before initiation of immunosuppressive treatment. Paired urine and blood samples were collected on the day of biopsy. Of the patients enrolled, 6 (8.7%) were diagnosed as focal class, 16 (23.2%) as crescentic class, 15 (21.7%) as sclerotic class and 32 (46.4%) as mixed class, according to the histological system proposed by Berden et al. [2]. Clinical features stratified per classification group are presented in Table 1. eGFR at the time of biopsy was found to be higher in the focal class versus all other classes. There was no difference among groups with respect to age, gender, body mass index and hypertension. Table 1. Characteristics of ANCA-GN patients by the renal histological class in the test set Focal Crescentic Mixed Sclerosis P-value (n = 6) (n = 16) (n = 32) (n = 15) Characteristics on admission  Age (years) 36 ± 7 47 ± 11 41 ± 13 47 ± 13 0.106  Men, n (%) 3 (50) 7 (43.7) 15 (46.8) 5 (33) 0.915  Hypertension, n (%) 1 (16.7) 1 (6.25) 4 (12.5) 2 (13.3) 0.881  BMI (kg/m2) 21.5 ± 2.4 21.9 ± 2.9 21.0 ± 2.4 20.5 ± 2.7 0.198 Characteristics at the time of biopsy  SCr (mg/dL) 0.9 ± 0.3 5.0 ± 2.7 4.1 ± 2.7 4.1 ± 2.0 0.001  eGFR (mL/min/1.73 m) 82.6 ± 28.7 16.4 ± 12.1 29.1 ± 22.7 24.1 ± 16.5 0.001  MPO-ANCA (IU/mL) 221.4 ± 97.5 249.9 ± 129.9 277.7 ± 146.5 371.6 ± 169.1 0.272  Ualb (mg/mg Cr) 0.8 ± 0.4 3.4 ± 1.9 2.1 ± 1.0 2.2 ± 1.3 0.001 Focal Crescentic Mixed Sclerosis P-value (n = 6) (n = 16) (n = 32) (n = 15) Characteristics on admission  Age (years) 36 ± 7 47 ± 11 41 ± 13 47 ± 13 0.106  Men, n (%) 3 (50) 7 (43.7) 15 (46.8) 5 (33) 0.915  Hypertension, n (%) 1 (16.7) 1 (6.25) 4 (12.5) 2 (13.3) 0.881  BMI (kg/m2) 21.5 ± 2.4 21.9 ± 2.9 21.0 ± 2.4 20.5 ± 2.7 0.198 Characteristics at the time of biopsy  SCr (mg/dL) 0.9 ± 0.3 5.0 ± 2.7 4.1 ± 2.7 4.1 ± 2.0 0.001  eGFR (mL/min/1.73 m) 82.6 ± 28.7 16.4 ± 12.1 29.1 ± 22.7 24.1 ± 16.5 0.001  MPO-ANCA (IU/mL) 221.4 ± 97.5 249.9 ± 129.9 277.7 ± 146.5 371.6 ± 169.1 0.272  Ualb (mg/mg Cr) 0.8 ± 0.4 3.4 ± 1.9 2.1 ± 1.0 2.2 ± 1.3 0.001 eGFR determined by CKD Epidemiology Collaboration (CKD-EPI) equation. Values presented as mean ± SD unless stated otherwise. CR, creatinine; SCr, serum creatinine. Table 1. Characteristics of ANCA-GN patients by the renal histological class in the test set Focal Crescentic Mixed Sclerosis P-value (n = 6) (n = 16) (n = 32) (n = 15) Characteristics on admission  Age (years) 36 ± 7 47 ± 11 41 ± 13 47 ± 13 0.106  Men, n (%) 3 (50) 7 (43.7) 15 (46.8) 5 (33) 0.915  Hypertension, n (%) 1 (16.7) 1 (6.25) 4 (12.5) 2 (13.3) 0.881  BMI (kg/m2) 21.5 ± 2.4 21.9 ± 2.9 21.0 ± 2.4 20.5 ± 2.7 0.198 Characteristics at the time of biopsy  SCr (mg/dL) 0.9 ± 0.3 5.0 ± 2.7 4.1 ± 2.7 4.1 ± 2.0 0.001  eGFR (mL/min/1.73 m) 82.6 ± 28.7 16.4 ± 12.1 29.1 ± 22.7 24.1 ± 16.5 0.001  MPO-ANCA (IU/mL) 221.4 ± 97.5 249.9 ± 129.9 277.7 ± 146.5 371.6 ± 169.1 0.272  Ualb (mg/mg Cr) 0.8 ± 0.4 3.4 ± 1.9 2.1 ± 1.0 2.2 ± 1.3 0.001 Focal Crescentic Mixed Sclerosis P-value (n = 6) (n = 16) (n = 32) (n = 15) Characteristics on admission  Age (years) 36 ± 7 47 ± 11 41 ± 13 47 ± 13 0.106  Men, n (%) 3 (50) 7 (43.7) 15 (46.8) 5 (33) 0.915  Hypertension, n (%) 1 (16.7) 1 (6.25) 4 (12.5) 2 (13.3) 0.881  BMI (kg/m2) 21.5 ± 2.4 21.9 ± 2.9 21.0 ± 2.4 20.5 ± 2.7 0.198 Characteristics at the time of biopsy  SCr (mg/dL) 0.9 ± 0.3 5.0 ± 2.7 4.1 ± 2.7 4.1 ± 2.0 0.001  eGFR (mL/min/1.73 m) 82.6 ± 28.7 16.4 ± 12.1 29.1 ± 22.7 24.1 ± 16.5 0.001  MPO-ANCA (IU/mL) 221.4 ± 97.5 249.9 ± 129.9 277.7 ± 146.5 371.6 ± 169.1 0.272  Ualb (mg/mg Cr) 0.8 ± 0.4 3.4 ± 1.9 2.1 ± 1.0 2.2 ± 1.3 0.001 eGFR determined by CKD Epidemiology Collaboration (CKD-EPI) equation. Values presented as mean ± SD unless stated otherwise. CR, creatinine; SCr, serum creatinine. Twenty-five ANCA-GN patients were included in the validation set and exhibited characteristics similar to those in the test set (Supplementary data, Table S1). We also included patients with proteinuria due to nonvasculitic glomerular diseases (FSGS and MN) and patients with impaired renal function due to nonglomerular diseases (ATN and CON). Clinical details of these patients are listed in Supplementary data, Table S2. In the second stage, UAGT, 24-h proteinuria and serum creatinine and MPO-ANCA were monitored in 50 subjects with ANCA-GN in the test set for up to 24 months (Figure 1). Urinary AGT level correlated with renal pathological activity in ANCA-GN In the test set, UAGT on the day of biopsy was determined in patients with ANCA-GN, in healthy volunteers and in patients with other renal diseases. Patients with ANCA-GN had significantly greater UAGT [median 355.27 ng/mg of creatinine (first and third quartiles, 158.42 and 614.85)] compared with healthy volunteers [1.39 ng/mg of creatinine (0.92 and 2.78)], patients with FSGS [44.57 ng/mg of creatinine (27.67 and 62.22)], patients with MN [55.86 ng/mg of creatinine (31.86 and 72.29)], patients with ATN [73.63 ng/mg of creatinine (49.44 and 93.36)] and patients with CON [87.56 ng/mg of creatinine (65.05 and 102.56)] (all P < 0.001; Supplementary data, Figure S1A). In contrast to urinalysis, serum AGT levels were comparable among groups (Supplementary data, Figure S1B). There was no correlation between serum and UAGT (r = 0.067, P = 0.387; Supplementary data, Figure S1C). In the test set, UAGT in ANCA-GN on the day of biopsy was compared with the histological parameters for active renal injury (cellular crescents, endocapillary proliferation, fibrinoid necrosis and interstitial inflammation) and parameters for chronic renal injury (glomerular sclerosis, fibrous crescents, tubular atrophy and interstitial fibrosis). As shown in Figure 2, UAGT closely correlated with the extent of active renal injury, particularly the percentages of glomeruli showing cellular crescents (r = 0.616, P < 0.001; Figure 2A) and the severity of acute interstitial inflammation (r = 0.502, P < 0.001; Figure 2B). The extent of chronic renal injury did not correlate with UAGT (Supplementary data, Table S3). FIGURE 2: View largeDownload slide UAGT serves as an indicator of active crescentic ANCA-GN in the test set. (A) Correlation between UAGT level at the time of biopsy and the percentage of glomeruli showing cellular crescents in ANCA-GN. (B) Correlation between UAGT level at the time of biopsy and the extent of acute interstitial inflammation in ANCA-GN. (C and D) Sixty-nine ANCA-GN patients were subgrouped into focal, crescentic, mix and sclerotic classes, following the histological system proposed by Berden. (C) Urinary and (D) serum AGT in patients with different classes of ANCA-GN at the time of biopsy and in healthy volunteers. (E) ROC curve analysis to determine the cutoff value for UAGT and Ualb with respect to diagnosis of crescentic ANCA-GN. (F) ROC curve analysis for the test and validation sets. *P<0.05 versus healthy volunteers in (C) and (D). FIGURE 2: View largeDownload slide UAGT serves as an indicator of active crescentic ANCA-GN in the test set. (A) Correlation between UAGT level at the time of biopsy and the percentage of glomeruli showing cellular crescents in ANCA-GN. (B) Correlation between UAGT level at the time of biopsy and the extent of acute interstitial inflammation in ANCA-GN. (C and D) Sixty-nine ANCA-GN patients were subgrouped into focal, crescentic, mix and sclerotic classes, following the histological system proposed by Berden. (C) Urinary and (D) serum AGT in patients with different classes of ANCA-GN at the time of biopsy and in healthy volunteers. (E) ROC curve analysis to determine the cutoff value for UAGT and Ualb with respect to diagnosis of crescentic ANCA-GN. (F) ROC curve analysis for the test and validation sets. *P<0.05 versus healthy volunteers in (C) and (D). UAGT as an indicator of crescentic ANCA-GN In the test set, UAGT was compared among different histological groups. In crescentic ANCA-GN, UAGT dramatically increased [1120.51 ng/mg of creatinine (first and third quartiles, 483.33 and 1828.08)] compared with mixed [325.46 ng/mg of creatinine (200.26 and 459.06)], sclerotic [202.35 ng/mg of creatinine (90.25 and 406.35)] or focal ANCA-GN [115.26 ng/mg of creatinine (60.71 and 272.58)] (all P < 0.001; Figure 2C). Serum AGT levels were comparable among different histological groups (Figure 2D). UAGT associated with the existence of crescentic ANCA-GN (Supplementary data, Figure S2). This association was supported by the multivariable analyses. After adjustment for clinical variables including age and eGFR on the day of biopsy, UAGT was the most powerful indicator of crescentic ANCA-GN. The highest quartile of UAGT on the day of biopsy was associated with an increased risk for crescentic ANCA-GN by 6-fold compared with the lowest quartile (Supplementary data, Table S4 and Figure S2). To assess the diagnostic value of UAGT for crescentic ANCA-GN, an ROC curve was generated. The AUC of UAGT on the day of biopsy for diagnosing crescentic ANCA-GN was 0.88, which was greater than those of existing clinical markers, such as urinary albumin:creatinine ratio (Ualb, 0.73, P = 0.029), eGFR (0.69, P = 0.009), MPO-ANCA (0.42, P < 0.001) and the combined clinical markers (0.79, P = 0.039) (Figure 2E and Supplementary data, Table S5). We further evaluated the performance of a combination of UAGT and the clinical markers in the test set. When UAGT was added to the clinical markers, the performance for identifying crescentic ANCA-GN was further improved (all P < 0.05) (Table 2). Table 2. Performance of a combination of UAGT and clinical markers for diagnosing crescentic ANCA-GN in the test set Clinical marker AUC (95%) Clinical marker Clinical marker and UAGT P-value Ualb 0.729 (0.591–0.868) 0.893 (0.789–0.996) 0.009 eGFR 0.690 (0.560–0.820) 0.890 (0.785–0.996) 0.001 MPO-ANCA 0.422 (0.255–0.590) 0.874 (0.755–0.993) 0.010 Combination of clinical markers 0.795 (0.681–0.883) 0.894 (0.796–0.955) 0.034 Clinical marker AUC (95%) Clinical marker Clinical marker and UAGT P-value Ualb 0.729 (0.591–0.868) 0.893 (0.789–0.996) 0.009 eGFR 0.690 (0.560–0.820) 0.890 (0.785–0.996) 0.001 MPO-ANCA 0.422 (0.255–0.590) 0.874 (0.755–0.993) 0.010 Combination of clinical markers 0.795 (0.681–0.883) 0.894 (0.796–0.955) 0.034 eGFR determined by CKD Epidemiology Collaboration (CKD-EPI) equation. Table 2. Performance of a combination of UAGT and clinical markers for diagnosing crescentic ANCA-GN in the test set Clinical marker AUC (95%) Clinical marker Clinical marker and UAGT P-value Ualb 0.729 (0.591–0.868) 0.893 (0.789–0.996) 0.009 eGFR 0.690 (0.560–0.820) 0.890 (0.785–0.996) 0.001 MPO-ANCA 0.422 (0.255–0.590) 0.874 (0.755–0.993) 0.010 Combination of clinical markers 0.795 (0.681–0.883) 0.894 (0.796–0.955) 0.034 Clinical marker AUC (95%) Clinical marker Clinical marker and UAGT P-value Ualb 0.729 (0.591–0.868) 0.893 (0.789–0.996) 0.009 eGFR 0.690 (0.560–0.820) 0.890 (0.785–0.996) 0.001 MPO-ANCA 0.422 (0.255–0.590) 0.874 (0.755–0.993) 0.010 Combination of clinical markers 0.795 (0.681–0.883) 0.894 (0.796–0.955) 0.034 eGFR determined by CKD Epidemiology Collaboration (CKD-EPI) equation. To validate the diagnostic value of UAGT for crescentic ANCA-GN, the AUC for identifying crescentic ANCA-GN was analyzed in an independent validation set. As demonstrated in Figure 2F, the ability of UAGT to diagnose crescentic ANCA-GN was validated in the validation set (AUC = 0.81). UAGT as a marker for treatment response and renal relapse Of the 69 ANCA-GN patients in the test set, 19 were excluded (9 died, 8 progressed to end-stage renal disease and 2 were lost to follow-up). Therefore, in the second stage, renal function and UAGT were monitored in 50 ANCA-GN patients for up to 24 months (Figure 1). All the patients received the standard induction treatment with cyclophosphamide and glucocorticoids or rituximab, followed by maintenance therapy with azathioprine and prednisolone. Renal relapse is defined as an initiation of immunosuppressive rescue therapy due to an increasing serum creatinine and an increasing number of urinary red cells [27]. As shown in Figure 3A, UAGT significantly decreased during immunosuppressive treatment and remained low during follow-up (P < 0.001). The reduction in UAGT was even more pronounced when patients with relapsing disease were excluded during follow-up (Figure 3B). FIGURE 3: View largeDownload slide UAGT levels in ANCA-GN during follow-up in the test set. (A) Urinary levels of AGT decreased during the initial immunosuppressive treatment and remained low during the maintenance therapy. The AGT measurements obtained at the time of biopsy (n = 69) and after 8 months (n = 64), 16 months (n = 46) and 24 months (n = 50) of follow-up. (B) UAGT measured during the 24-month follow-up excluding patients with relapsing disease (n = 62 at the time of biopsy, n = 57 after 8 months, n = 39 after 16 months and n = 43 after 24 months). (C–E) Longitudinal study with measurements of UAGT in patients suffering from renal relapse. Arrows indicate the onset of relapse. *P < 0.05 in (A) and (B). FIGURE 3: View largeDownload slide UAGT levels in ANCA-GN during follow-up in the test set. (A) Urinary levels of AGT decreased during the initial immunosuppressive treatment and remained low during the maintenance therapy. The AGT measurements obtained at the time of biopsy (n = 69) and after 8 months (n = 64), 16 months (n = 46) and 24 months (n = 50) of follow-up. (B) UAGT measured during the 24-month follow-up excluding patients with relapsing disease (n = 62 at the time of biopsy, n = 57 after 8 months, n = 39 after 16 months and n = 43 after 24 months). (C–E) Longitudinal study with measurements of UAGT in patients suffering from renal relapse. Arrows indicate the onset of relapse. *P < 0.05 in (A) and (B). Among 50 patients who were followed up for 24 months, 7 of them suffered from relapsing ANCA-GN. In three of the seven patients, UAGT was assessed at the onset of relapse. The renal relapse was associated with an elevation in UAGT (Figure 3C–E). The corresponding UAGT, 24-h proteinuria, serum creatinine and MPO-ANCA are presented in Supplementary data, Table S6. UAGT reflects intrarenal RAS status To investigate the relationship between UAGT and renal production of AGT, immunohistochemistry of AGT and angiotensin II was performed in renal biopsies from ANCA-GN patients. Immunoreactivity of AGT and angiotensin II significantly increased in biopsies of crescentic ANCA-GN compared with that in normal kidneys, and in biopsies of mixed, sclerotic or focal ANCA-GN (all P < 0.001; Figure 4). Overexpression of AGT in biopsies was observed predominantly in proximal tubules and cellular crescents (Figure 4A). Staining of angiotensin II was detected mainly in distal tubules and cellular crescents (Figure 4A). Glomerular and tubular expression of AGT or angiotensin II positively correlated with UAGT on the day of biopsy (Figure 4C, E). FIGURE 4: View largeDownload slide Expression of intrarenal RAS correlates with UAGT level in patients with ANCA-GN. (A) Representative photos of intrarenal AGT and angiotensin II (Ang II) expression in glomeruli and tubulointerstitium shown by immunohistochemical staining. (B) Semiquantitative data of intrarenal AGT staining. (C) Correlation between glomerular or tubulointerstitial AGT expression and UAGT levels. (D) Semiquantitative data of intrarenal Ang II staining. (E) Correlation between glomerular or tubulointerstitial Ang II expression and UAGT levels. Scale bar = 100 µm. *P < 0.05 versus normal kidney in (B) and (D). FIGURE 4: View largeDownload slide Expression of intrarenal RAS correlates with UAGT level in patients with ANCA-GN. (A) Representative photos of intrarenal AGT and angiotensin II (Ang II) expression in glomeruli and tubulointerstitium shown by immunohistochemical staining. (B) Semiquantitative data of intrarenal AGT staining. (C) Correlation between glomerular or tubulointerstitial AGT expression and UAGT levels. (D) Semiquantitative data of intrarenal Ang II staining. (E) Correlation between glomerular or tubulointerstitial Ang II expression and UAGT levels. Scale bar = 100 µm. *P < 0.05 versus normal kidney in (B) and (D). To localize AGT-expressing cells in the kidney, double staining was performed in biopsies of patients with crescentic ANCA-GN. Overexpression of AGT was localized mainly in proximal tubular cells, infiltrated macrophages, podocytes and parietal epithelial cells (Figure 5). FIGURE 5: View largeDownload slide Overexpression of AGT in crescentic ANCA-GN was localized mainly in proximal tubular cells, infiltrated macrophages, podocytes and parietal epithelial cells. Representative photographs of AGT localization determined with double staining of antibody against AGT and antibodies against markers of renal tubular segments, markers of macrophages, markers of podocytes or markers of parietal epithelial cells. Scale bar = 100 µm. AQP1, aquaporin 1 (proximal tubule); THP, Tamm–Horsfall protein (thick ascending limb); AQP2, NCCT or thiazide-sensitive NaCl cotransporter (distal tubule); aquaporin 2 (collecting duct); CD68 (macrophage); WT1, Wilms’ tumor (podocyte); Claudin 1 (parietal epithelial cell). FIGURE 5: View largeDownload slide Overexpression of AGT in crescentic ANCA-GN was localized mainly in proximal tubular cells, infiltrated macrophages, podocytes and parietal epithelial cells. Representative photographs of AGT localization determined with double staining of antibody against AGT and antibodies against markers of renal tubular segments, markers of macrophages, markers of podocytes or markers of parietal epithelial cells. Scale bar = 100 µm. AQP1, aquaporin 1 (proximal tubule); THP, Tamm–Horsfall protein (thick ascending limb); AQP2, NCCT or thiazide-sensitive NaCl cotransporter (distal tubule); aquaporin 2 (collecting duct); CD68 (macrophage); WT1, Wilms’ tumor (podocyte); Claudin 1 (parietal epithelial cell). DISCUSSION One of the major challenges in optimizing management of ANCA-GN is the lack of a high-performance indicator to ascertain histological lesions and disease activity. Currently assessment of renal injury can be only achieved in kidney biopsies, which is obtained through an invasive procedure [2, 8, 28]. Thus it is difficult to dynamically monitor renal disease activity, which may allow for evaluation of therapy efficacy and renal relapse in clinical circumstances. In the present study, we demonstrate that UAGT paralleled kidney RAS status and renal pathological activity in ANCA-GN. Tracking UAGT could be used to monitor treatment response and renal relapse over time. In the first phase of the study, we found that UAGT, measured on the day of the biopsy, varied significantly depending on the renal pathological activity in ANCA-GN. Recent studies have demonstrated that UAGT reflects the severity of renal histology [18, 19]. However, association between UAGT and renal pathological activity in ANCA-GN has not been established. Our data showed that UAGT at the time of biopsy strongly correlated with the number of cellular crescents and the degree of interstitial inflammation. Such an association between UAGT and active renal damage suggested a potential role of UAGT in identifying active ANCA-GN. This usefulness of UAGT would not be affected by renal function impairment, proteinuria or hypertension. Patients with impaired renal function (i.e. ATN/CON) or sustained proteinuria (i.e. FSGS/MN) exhibited much lower UAGT than those with ANCA-GN. In patients with ANCA-GN, UAGT did not correlated with systolic (r = 0.12, P = 0.318) or diastolic blood pressure (r = 0.13, P = 0.269). More importantly, we demonstrated that UAGT is a novel biomarker for crescentic ANCA-GN. Based on the histological classification schema proposed by Berden et al. [2], ANCA-GN has been classified into four distinct categories: focal, crescentic, mixed and sclerotic. Crescentic ANCA-GN is a particularly aggressive type of ANCA-GN that associated with highly active renal lesions, rapidly declining renal function and a good chance of renal function recovery [2, 10, 29–31]. UAGT was markedly elevated in patients with crescentic ANCA-GN. The performance of UAGT for identifying crescentic ANCN-GN is superior to other existing clinical screening markers such as Ualb, eGFR, MPO-ANCA and the combined clinical markers. Furthermore, we demonstrated that the addition of UAGT to existing clinical markers significantly increased the accuracy of diagnosing crescentic ANCA-GN, as demonstrated by AUC results. The value of UAGT for identifying crescentic ANCA-GN was further demonstrated in a validation cohort. These results support the use of UAGT, especially the combination use of UAGT with other clinical markers, in identifying crescentic ANCA-GN. A large number of clinical studies demonstrate that the presence of specific histological lesions in renal biopsies is helpful in predicting renal outcome and in planning therapy [2, 31–33]. Active glomerular lesions, in particular cellular crescents formation, have been found to correlate with recovery of renal function irrespective of baseline GFR [2, 31]. Therefore, measurement of UAGT is of great help in identifying patients with active renal damage, in particular crescentic ANCA-GN. Timely recognition of active renal involvement would prompt consideration of immunosuppressive treatment to prevent irreversible kidney damage. Monitoring of renal disease activity could be beneficial in clinical practice. Identifying patients with relapsing and persistent disease would help physicians to optimize management and minimize treatment-related side effects [5, 24]. In the second phase of the study, we evaluated the value of UAGT in the monitoring of renal disease activity in ANCA-GN. UAGT generally decreased after the induction of immunotherapy and stayed low during 24 months of follow-up. Patients experiencing renal relapses exhibited an elevation in UAGT. Therefore it is conceivable to speculate that UAGT reflects disease activity and can serve as an indicator of renal relapse in ANCA-GN. Supporting our findings, a previous report demonstrated a reduction in UAGT following steroids therapy in patients with chronic GN [17]. However, whether UAGT is predictive of relapse remains unclear and needs further studies. Both urinary and serum AGT were detected simultaneously in this study. Contrary to UAGT, serum AGT was not elevated in ANCA-GN. Thus UAGT may not reflect systemic RAS status. A potential site of AGT secretion into the urine is the kidney [19]. In the present study, immunohistochemistry analysis revealed a significantly increased expression of AGT and angiotensin II in renal biopsies from patients with crescentic ANCA-GN. UAGT correlated well with intrarenal expression of AGT and angiotensin II, suggesting that increased UAGT excretion in ANCA-GN may be due to enhanced synthesis in the kidney. Consistently, staining of AGT, an important substrate of angiotensin II, has been identified in tubular cells, macrophages, parietal epithelial cells and podocytes, which makes its secretion to the urine possible. Increased production of intrarenal AGT under pathological conditions can be mediated through positive feedback from angiotensin II [34]. As such, UAGT might also be proposed as a marker of intrarenal RAS status. This extends similar findings in patients with chronic kidney disease [11, 17], type 2 diabetes [35], hypertension [36], polycystic disease [37] as well as podocyte injury [38]. Intrarenal RAS has been identified as a key pathogenic mediator of cell proliferation and migration that results in renal inflammation and crescent formation [11–13]. In our study, we demonstrated significantly increased expression of intrarenal RAS that positively correlated with UAGT in crescentic ANCA-GN. This result provides a rationale to use angiotensin receptor blockers to reduce active renal tissue injury in patients with ANCA-GN. Indeed, RAS inhibitor has been demonstrated to improve renal function and induce regression of crescentic lesions in patients with crescentic ANCA-GN [12]. Our present study has the following strengths. First, all subjects enrolled had a concomitant renal biopsy performed before initiation of immunosuppressive treatment. Paired urine and blood samples were collected on the day of biopsy, allowing us to evaluate the correlation between UAGT and renal pathological activity or intrarenal RAS status. Double immunofluorescent staining identified proximal tubules, macrophages, podocytes and parietal epithelium expressing AGT in the kidney. Additionally, we compared the diagnostic performance of UAGT with existing clinical makers in crescentic ANCA-GN. We demonstrated that UAGT is a powerful indicator of crescentic ANCA-GN and outperforms the existing clinical markers. This result has been confirmed in a validation cohort. Finally, we followed the subjects with ANCA-GN every 4 months for 24 months after biopsy, which allowed us to assess the ability of UAGT in indicating disease activity and renal relapse in ANCA-GN. This study also has some limitations. First, only patients not being treated with RAS inhibitors were enrolled, therefore the influence of RAS blockade on the value of UAGT as a biomarker of disease activity remains to be investigated in our ongoing serial studies. Second, we did not include patients with active nonrenal ANCA-associated vasculitis and thus cannot exclude the contribution of AGT from the circulation into the urine. However, serum AGT was not elevated in ANCA-GN patients and did not correlated with UAGT. Therefore it seems unlikely that the increased UAGT in ANCA-GN was mainly due to filtered AGT from the systemic circulation. In addition, urinary, instead of serum AGT, serves as an indicator of renal disease activity. In clinical settings there are circumstances, however, in which urine samples are unavailable (i.e. patients with oliguria). Moreover, all ANCA-GN patients included were MPO-ANCA positive. Since there is a striking predominance of MPO-ANCA over proteinase 3 (PR3)-ANCA in Chinese people [39], validation of our findings in cohorts with enough patients with PR3-ANCA-GN would be desirable. In conclusion, our study shows that UAGT could serve as a useful biomarker for renal pathology activity and could be used to monitor treatment response and renal relapse. Application of this novel biomarker clinically would allow for evaluation of therapy efficacy and minimization of treatment-related toxicity in ANCA-GN. SUPPLEMENTARY DATA Supplementary data are available at ndt online. FUNDING This work was supported by the National Natural Science Foundation of China (81570619). AUTHORS’ CONTRIBUTIONS L.W. performed the experiments and evaluated and interpreted data. M.Y., L.J. and X.F. performed histological and biochemical experiments, evaluated data and contributed to manuscript preparation. Z.Z., M.Y., X.Z., M.S., S.C., C.W. and Z.Y. performed biochemical experiments and contributed to manuscript preparation. S.C. and C.W. helped to enroll patients in the validation set. W.C. designed, supervised and financed the study and wrote the manuscript. All authors have reviewed and revised the manuscript. CONFLICT OF INTEREST STATEMENT None declared. REFERENCES 1 Jennette JC , Falk RJ. Small-vessel vasculitis . N Engl J Med 1997 ; 337 : 1512 – 1523 Google Scholar CrossRef Search ADS PubMed 2 Berden AE , Ferrario F , Hagen EC et al. Histopathologic classification of ANCA-associated glomerulonephritis . J Am Soc Nephrol 2010 ; 21 : 1628 – 1636 Google Scholar CrossRef Search ADS PubMed 3 Jones RB , Tervaert JW , Hauser T et al. Rituximab versus cyclophosphamide in ANCA-associated renal vasculitis . N Engl J Med 2010 ; 363 : 211 – 220 Google Scholar CrossRef Search ADS PubMed 4 Specks U , Merkel PA , Seo P et al. Efficacy of remission-induction regimens for ANCA-associated vasculitis . N Engl J Med 2013 ; 369 : 417 – 427 Google Scholar CrossRef Search ADS PubMed 5 Schonermarck U , Gross WL , de Groot K. Treatment of ANCA-associated vasculitis . Nat Rev Nephrol 2014 ; 10 : 25 – 36 Google Scholar CrossRef Search ADS PubMed 6 Little MA , Nightingale P , Verburgh CA et al. Early mortality in systemic vasculitis: relative contribution of adverse events and active vasculitis . Ann Rheum Dis 2010 ; 69 : 1036 – 1043 Google Scholar CrossRef Search ADS PubMed 7 Falk RJ , Jennette JC. ANCA small-vessel vasculitis . J Am Soc Nephrol 1997 ; 8 : 314 – 322 Google Scholar PubMed 8 Rahmattulla C , Bruijn JA , Bajema IM. Histopathological classification of antineutrophil cytoplasmic antibody-associated glomerulonephritis. An update . Curr Opin Nephrol Hypertens 2014 ; 23 : 224 – 231 Google Scholar CrossRef Search ADS PubMed 9 Jennette JC , Falk RJ. Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease . Nat Rev Rheumatol 2014 ; 10 : 463 – 473 Google Scholar CrossRef Search ADS PubMed 10 Singh SK , Jeansson M , Quaggin SE. New insights into the pathogenesis of cellular crescents . Curr Opin Nephrol Hypertens 2011 ; 20 : 258 – 262 Google Scholar CrossRef Search ADS PubMed 11 Kobori H , Nangaku M , Navar LG et al. The intrarenal renin–angiotensin system: from physiology to the pathobiology of hypertension and kidney disease . Pharmacol Rev 2007 ; 59 : 251 – 287 Google Scholar CrossRef Search ADS PubMed 12 Rizzo P , Perico N , Gagliardini E et al. Nature and mediators of parietal epithelial cell activation in glomerulonephritides of human and rat . Am J Pathol 2013 ; 183 : 1769 – 1778 Google Scholar CrossRef Search ADS PubMed 13 Aki K , Shimizu A , Masuda Y et al. ANG II receptor blockade enhances anti-inflammatory macrophages in anti-glomerular basement membrane glomerulonephritis . Am J Physiol Renal Physiol 2010 ; 298 : F870 – F882 Google Scholar CrossRef Search ADS PubMed 14 Urushihara M , Ohashi N , Miyata K et al. Addition of angiotensin II type 1 receptor blocker to CCR2 antagonist markedly attenuates crescentic glomerulonephritis . Hypertension 2011 ; 57 : 586 – 593 Google Scholar CrossRef Search ADS PubMed 15 Kinoshita Y , Kondo S , Urushihara M et al. Angiotensin II type I receptor blockade suppresses glomerular renin–angiotensin system activation, oxidative stress, and progressive glomerular injury in rat anti-glomerular basement membrane glomerulonephritis . Transl Res 2011 ; 158 : 235 – 248 Google Scholar CrossRef Search ADS PubMed 16 Minutolo R , Balletta MM , Catapano F et al. Mesangial hypercellularity predicts antiproteinuric response to dual blockade of RAS in primary glomerulonephritis . Kidney Int 2006 ; 70 : 1170 – 1176 Google Scholar CrossRef Search ADS PubMed 17 Urushihara M , Kondo S , Kagami S et al. Urinary angiotensinogen accurately reflects intrarenal renin–angiotensin system activity . Am J Nephrol 2010 ; 31 : 318 – 325 Google Scholar CrossRef Search ADS PubMed 18 Kim SM , Jang HR , Lee YJ et al. Urinary angiotensinogen levels reflect the severity of renal histopathology in patients with chronic kidney disease . Clin Nephrol 2011 ; 76 : 117 – 123 Google Scholar CrossRef Search ADS PubMed 19 Yamamoto T , Nakagawa T , Suzuki H et al. Urinary angiotensinogen as a marker of intrarenal angiotensin II activity associated with deterioration of renal function in patients with chronic kidney disease . J Am Soc Nephrol 2007 ; 18 : 1558 – 1565 Google Scholar CrossRef Search ADS PubMed 20 Cao W , Jin L , Zhou Z et al. Overexpression of intrarenal renin–angiotensin system in human acute tubular necrosis . Kidney Blood Press Res 2016 ; 41 : 746 – 756 Google Scholar CrossRef Search ADS PubMed 21 Kobori H , Harrison-Bernard LM , Navar LG. Urinary excretion of angiotensinogen reflects intrarenal angiotensinogen production . Kidney Int 2002 ; 61 : 579 – 585 Google Scholar CrossRef Search ADS PubMed 22 Kobori H , Katsurada A , Ozawa Y et al. Enhanced intrarenal oxidative stress and angiotensinogen in IgA nephropathy patients . Biochem Biophys Res Commun 2007 ; 358 : 156 – 163 Google Scholar CrossRef Search ADS PubMed 23 Kobori H , Ohashi N , Katsurada A et al. Urinary angiotensinogen as a potential biomarker of severity of chronic kidney diseases . J Am Soc Hypertens 2008 ; 2 : 349 – 354 Google Scholar CrossRef Search ADS PubMed 24 Moroni G , Ponticelli C. Rapidly progressive crescentic glomerulonephritis: early treatment is a must . Autoimmun Rev 2014 ; 13 : 723 – 729 Google Scholar CrossRef Search ADS PubMed 25 DeLong ER , DeLong DM , Clarke-Pearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach . Biometrics 1988 ; 44 : 837 – 845 Google Scholar CrossRef Search ADS PubMed 26 Shrout PE , Fleiss JL. Intraclass correlations: uses in assessing rater reliability . Psychol Bull 1979 ; 86 : 420 – 428 Google Scholar CrossRef Search ADS PubMed 27 Brix SR , Stege G , Disteldorf E et al. CC chemokine ligand 18 in ANCA-associated crescentic GN . J Am Soc Nephrol 2015 ; 26 : 2105 – 2117 Google Scholar CrossRef Search ADS PubMed 28 de Lind van Wijngaarden RA , Hauer HA , Wolterbeek R et al. Chances of renal recovery for dialysis-dependent ANCA-associated glomerulonephritis . J Am Soc Nephrol 2007 ; 18 : 2189 – 2197 Google Scholar CrossRef Search ADS PubMed 29 Iwakiri T , Fujimoto S , Kitagawa K et al. Validation of a newly proposed histopathological classification in Japanese patients with anti-neutrophil cytoplasmic antibody-associated glomerulonephritis . BMC Nephrol 2013 ; 14 : 125 Google Scholar CrossRef Search ADS PubMed 30 Lionaki S , Mavragani CP , Karras A et al. Predictors of renal histopathology in antineutrophil cytoplasmic antibody associated glomerulonephritis . J Autoimmun 2016 ; 72 : 57 – 64 Google Scholar CrossRef Search ADS PubMed 31 Hauer HA , Bajema IM , Van Houwelingen HC et al. Determinants of outcome in ANCA-associated glomerulonephritis: a prospective clinico-histopathological analysis of 96 patients . Kidney Int 2002 ; 62 : 1732 – 1742 Google Scholar CrossRef Search ADS PubMed 32 de Lind van Wijngaarden RA , Hauer HA , Wolterbeek R et al. Clinical and histologic determinants of renal outcome in ANCA-associated vasculitis: a prospective analysis of 100 patients with severe renal involvement . J Am Soc Nephrol 2006 ; 17 : 2264 – 2274 Google Scholar CrossRef Search ADS PubMed 33 Neumann I , Kain R , Regele H et al. Histological and clinical predictors of early and late renal outcome in ANCA-associated vasculitis . Nephrol Dial Transplant 2005 ; 20 : 96 – 104 Google Scholar CrossRef Search ADS PubMed 34 Kobori H , Ozawa Y , Suzaki Y et al. Young scholars award lecture: intratubular angiotensinogen in hypertension and kidney diseases . Am J Hypertens 2006 ; 19 : 541 – 550 Google Scholar CrossRef Search ADS PubMed 35 Persson F , Lu X , Rossing P et al. Urinary renin and angiotensinogen in type 2 diabetes: added value beyond urinary albumin? J Hypertens 2013 ; 31 : 1646 – 1652 Google Scholar CrossRef Search ADS PubMed 36 Michel FS , Norton GR , Maseko MJ et al. Urinary angiotensinogen excretion is associated with blood pressure independent of the circulating renin–angiotensin system in a group of African ancestry . Hypertension 2014 ; 64 : 149 – 156 Google Scholar CrossRef Search ADS PubMed 37 Salih M , Bovee DM , Roksnoer LCW et al. Urinary renin–angiotensin markers in polycystic kidney disease . Am J Physiol Renal Physiol 2017 ; 313 : F874 – F881 Google Scholar CrossRef Search ADS PubMed 38 Eriguchi M , Yotsueda R , Torisu K et al. Assessment of urinary angiotensinogen as a marker of podocyte injury in proteinuric nephropathies . Am J Physiol Renal Physiol 2016 ; 310 : F322 – F333 Google Scholar CrossRef Search ADS PubMed 39 Liu LJ , Chen M , Yu F et al. Evaluation of a new algorithm in classification of systemic vasculitis . Rheumatology (Oxford) 2008 ; 47 : 708 – 712 Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Nephrology Dialysis TransplantationOxford University Press

Published: May 4, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off