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Clinical and epidemiological research
Urinary epidermal growth factor predicts renal prognosis in antineutrophil cytoplasmic antibody-associated vasculitis
  1. Liang Wu1,2,3,4,
  2. Xiao-Qian Li1,2,3,4,
  3. Tanvi Goyal5,
  4. Sean Eddy5,
  5. Matthias Kretzler5,6,
  6. Wen-Jun Ju5,6,
  7. Min Chen1,2,3,4,
  8. Ming-Hui Zhao1,2,3,4
  1. 1 Department of Medicine, Renal Division, Peking University First Hospital, Beijing, China
  2. 2 Institute of Nephrology, Peking University, Beijing, China
  3. 3 Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, China
  4. 4 Key Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education, Beijing, China
  5. 5 Department of Internal Medicine, Division of Nephrology, University of Michigan, Ann Arbor, Michigan, USA
  6. 6 Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA
  1. Correspondence to Professor Min Chen, Renal Division, Peking University First Hospital, Peking University Institute of Nephrology, Beijing 100034, China; chenmin74{at}sina.com

Abstract

Introduction The current study aimed to investigate the association between urinary epidermal growth factor (uEGF) and renal disease severity and outcomes in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV).

Methods Intrarenal EGFmRNA expression was extracted from transcriptomic data of microdissected tubulointerstitial compartments of kidney biopsies of patients with AAV. uEGF was measured in 173 patients with AAV in active stage and 143 in remission, and normalised to urine creatinine excretion (uEGF/Cr). The association between uEGF/Cr (or EGFmRNA) and clinical–pathological parameters was tested using linear regression analysis. The ability of uEGF/Cr to predict renal outcomes was analysed using Cox’s regression analysis.

Results In patients with AAV, intrarenal EGFmRNA expression was significantly associated with estimated glomerular filtration rate (eGFR)(log2) at time of biopsy (β=0.63, p<0.001). The level of uEGF/Cr was significantly higher in patients in remission than in patients with active disease, both when looking at patients with sequential measurements (2.75±1.03vs 2.08±0.98, p<0.001) and in cross-sectional comparison. uEGF/Cr level was positively associated with eGFR(log2) at time of sampling in both active and remission stage (β=0.60, p<0.001; β=0.74, p<0.001, respectively). Patients with resistant renal disease had significantly lower uEGF/Cr levels than responders (1.65±1.22vs 2.16±1.26, p=0.04). Moreover, after adjusting for other potential predictors, uEGF/Cr was independently associated with composite endpoint of end-stage renal disease or 30% reduction of eGFR (HR 0.61, 95% CI 0.45 to 0.83, p=0.001).

Conclusion Lower uEGF/Cr levels are associated with more severe renal disease, renal resistance to treatment and higher risk of progression to composite outcome in patients with AAV.

  • autoimmune diseases
  • cytokines
  • autoantibodies

https://doi.org/10.1136/annrheumdis-2017-212578

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    Introduction

    Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of life-threatening autoimmune diseases, comprising microscopic polyangiitis, granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis.1 The kidney is one of the most commonly involved organs in AAV. With the initiation of immunosuppressive therapy, the prognosis of AAV has been improved greatly. However, approximately 25% of patients with AAV are still resistant to the induction therapy and 20%–25% of patients with AAV progress to end-stage renal disease (ESRD).2 3 It is therefore imperative to find non-invasive biomarkers predicting renal response to treatment and the deterioration of renal function in patients with AAV.

    Epidermal growth factor (EGF) is a peptide growth factor. Several studies have shown that urinary EGF (uEGF) is decreased in a variety of renal diseases including diabetic nephropathy,4 acute kidney injury,5 IgA nephropathy (IgAN),6 congenital ureteropelvic junction obstruction7 and chronic kidney disease (CKD) in children.8 More importantly, it has been shown that uEGF has the ability to predict the development of diabetic nephropathy9 and IgAN.6 In the present study, we investigated the association between intrarenal EGF mRNA and kidney function, and the association between uEGF and renal outcomes, including the renal response to treatment and renal disease progression in patients with AAV.

    Materials and methods

    Tubulointerstitial EGF mRNA expression in patients with AAV

    Human renal biopsies were collected in a multicentre study, the European Renal cDNA Bank (ERCB), after informed consent was obtained, according to the guidelines of the respective local ethics committees. Transcriptome analysis was performed on microdissected tubulointerstitial compartments of kidney biopsies of patients with AAV procured for molecular analysis from ERCB. The healthy controls biopsies were from living donor transplant biopsies. The details of biopsies handling have been described elsewhere.10 11 Briefly, immediately after renal biopsy, a minimum of 10% of the renal biopsy specimen was separated and stored in RNA preservative. Under a stereomicroscope, specimens were divided into glomeruli and tubulointerstitial compartments by manual microdissection. RNA was isolated following standard protocols. Tubulointerstitial EGF mRNA expression data were extracted from profiles of patients with AAV (table 1). Samples were hybridised on Affymetrix GeneChip U133 plus 2.12 Normalised expression data were log2-transformed.

    View this table:
    Table 1

    Basic demographic data and clinical information for ERCB patients whose intrarenal EGF mRNA level are available

    Patients and urine samples

    One hundred and seventy-three patients with active AAV, diagnosed from January 2001 to December 2016, were enrolled in this study. Urine samples from these patients were collected on the day of renal biopsy and before commencement of immunosuppressive treatment. Urine samples from 143 patients with AAV, who achieved complete or partial remission after immunosuppressive therapy (see definitions below), were collected at their regular ambulatory visits. Among the aforementioned patients with AAV, there were 60 patients who had urine samples of both active and remission stage. Altogether, 256 patients with AAV were enrolled in this study. All these patients were diagnosed at Peking University First Hospital and met the Chapel Hill Consensus Conference criteria of AAV.1 Patients with secondary vasculitis or other coexisting renal diseases, such as antiglomerular basement membrane nephritis, lupus nephritis and IgA nephropathy, were excluded. We also acquired 27 urine samples of age-matched and gender-matched healthy donors. The urine samples of all participants were centrifuged at 600 g for 5 min and stored at −80°C until tested. The research was done in compliance with the Declaration of Helsinki and approved by the ethics committee of our hospital. Written informed consent was obtained from each participant.

    Treatment

    The protocols of induction and maintenance treatment were described previously.13–15 More details are provided in the online supplementary text.

    Supplemental material

    Clinical evaluation

    Disease activity was assessed by the Birmingham Vasculitis Activity Score (BVAS).16 Remission was defined as the “absence of disease activity attributable to active disease qualified by the need for ongoing stable maintenance immunosuppressive therapy (complete remission)” or “at least 50% reduction of disease activity score and absence of new manifestations (partial remission)”. Treatment resistance was defined as unchanged or increased disease activity in patients with acute AAV after 4 weeks of treatment with standard therapy or a reduction of less than 50% in the disease activity score after 6 weeks of treatment, or chronic persistent disease defined as the presence of at least one major item or three minor items on the disease activity score list after >12 weeks of treatment.17

    The renal response to treatment, evaluated after initiation of immunosuppressive therapy, was judged according to the following criteria, as described previously18–20: (1) complete recovery of renal function was indicated by normalisation of renal function and resolution of haematuria; (2) partial recovery of renal function was indicated by stabilisation or improvement of renal function, with serum creatinine ≥133 μmol/L but dialysis-independent; and (3) treatment failure was indicated by progressive decline in kidney function with persistence of active urinary sediment despite immunosuppressive therapy.

    The estimated glomerular filtration rate (eGFR) was calculated using the adaptation of the four-factor Modification of Diet in Renal Disease study equation formula.21

    Detection of uEGF

    uEGF concentration was measured with Human EGF Immunoassay Quantikine ELISA according to the manufacturer’s instructions (R&D Systems). Samples (1:100 dilution for disease samples, 1:150 dilution for healthy control samples) and standards were run in duplicate. Meanwhile, three quality controls with high, medium and low concentrations using Quantikine Immunoassay Control Group 4 (R&D Systems) were included in each plate to control for interplate variance. Absorbance was read at 450 nm with the ELISA plate reader, and results were calculated with ELISACalc software. The concentration of the quality controls measured was in the acceptable ranges and interplate coefficients of variation of the quality controls were less than 15%. Urine creatinine was measured by kinetic Jaffe reaction using Quantikine Creatinine Assay Kit (R&D Systems). Finally, the level of uEGF was normalised to urine creatinine excretion, referred to uEGF/Cr. Log2-transformed uEGF/Cr value was used for all analyses.

    Renal histopathology

    The renal biopsy specimens were routinely examined by light microscopy, direct immunofluorescence and electron microscopy. Biopsies were independently scored by two pathologists blinded to the clinical data, according to the previously described standardised protocol for scoring renal biopsies of patients with AAV,22–24 with some minor modifications. The presence of crescents, sclerotic glomeruli and fibrinoid necrosis were calculated as the percentage of the total number of glomeruli in the biopsy. The severity of interstitial fibrosis and tubular atrophy (IFTA) was evaluated semiquantitatively and scored on a scale from 0 to 3: grade 0, no IFTA; grade 1, <25%; grade 2, 25–50%; and grade 3, >50%.25

    Outcome measurement

    Two parameters were employed to evaluate renal progression of AAV26: (1) the eGFR slope and (2) a composite endpoint of ESRD or 30% reduction of eGFR from the beginning of maintenance therapy. To calculate the eGFR slope over time, we fitted linear mixed-effects model to the outpatient eGFR records at the beginning of maintenance therapy for patients followed up for more than 12 months and had at least three eGFR records in remission stage. The endpoint events of patients were collected before 31 December 2017.

    Statistical analysis

    The continuous data were expressed as mean±SD or median and IQR. Categorical data were described by absolute frequencies and percentages. Comparison of clinical characteristic parameters between 173 patients in active stage and 143 patients in remission was assessed using a linear mixed model, with group as fixed effect, patients as a random effect and age, gender and other clinical parameters as dependent variables, respectively. A linear mixed model was also used to compare repeated measurements in 60 paired data. The model included uEGF/Cr as a dependent variable, group as a fixed effect and patient as a random effect. eGFR was added as covariate for eGFR adjustment. To compare independent continuous variables, Student’s t-test or Mann-Whitney U test was used as appropriate. Differences of qualitative results were compared using χ2 test. Linear regression analysis with unstandardised regression coefficients (β) as effect measure was performed to analyse the association between uEGF/Cr (or EGF mRNA) and other clinical parameters, in which uEGF/Cr (or EGF mRNA) acted as dependent variable, and eGFR (log2 transformed), IFTA grade, BVAS, C reactive protein (CRP) as well as eGFR slope acted as independent variables, respectively.

    The association between potential risk variables and the composite endpoint were analysed using Cox proportional hazards regression, first with one potential predictor at a time, second including all the potential predictors simultaneously. Patients with missing data were excluded from the analysis. Time-dependent receiver operating characteristic (ROC) curve analysis was conducted to evaluate the prognostic value of uEGF/Cr for the composite endpoint. In addition, we examined nested Cox proportional hazards models to evaluate the additive effect of uEGF/Cr on traditional predictive markers for renal progression. The goodness of fit and improved prediction ability of the additional parameters was assessed by R2, likelihood ratio tests and Akaike information criterion. Analysis was performed with SPSS statistical software package (SPSS V.24.0; IBM) and SAS (V.9.3). Differences were considered significant if p value <0.05.

    Results

    Intrarenal EGF mRNA in patients with AAV compared with living donors and association with eGFR

    To investigate whether EGF is associated with kidney impairment, we extracted intrarenal EGF mRNA expression data in samples from patients with AAV from the ERCB (table 1) and from healthy living donors. Figure 1 illustrates reduced EGF steady-state mRNA levels in the tubulointerstitial compartment in samples of patients with AAV relative to healthy living donor samples (7.85±1.17 vs 9.53±0.65, p<0.001, by Student’s t-test). In addition, linear regression analysis showed that tubulointerstitial EGF mRNA level was significantly associated with eGFR(log2) at time of biopsy (β=0.63, p<0.001).

    Figure 1
    Figure 1

    EGF mRNA is significantly lower in patients with AAV (n=21) in comparison with controls (n=18). Horizontal lines represent mean±SD. AAV, antineutrophil cytoplasmic antibody-associated vasculitis; EGF, epidermal growth factor.

    The levels of uEGF/Cr between patients with active disease and patients in remission

    The general data of patients with AAV are listed in table 2. For patients who had only one urine sample collection (113 patients in active stage and 83 patients in remission), the level of uEGF/Cr in active stage was significantly lower than that in remission (2.04±1.41 vs 2.63±1.31, p<0.001, by Student’s t-test) and was also significantly lower than healthy controls (2.04±1.41 vs 4.34±0.76, p<0.001, by Student’s t-test) (figure 2A).

    Figure 2
    Figure 2

    uEGF/Cr levels in patients with AAV. (A) The levels of urinary EGF/Cr(log2) in active stage, in remission and normal controls. Horizontal lines represent mean±SD. (B) Change of uEGF/Cr levels in 60 patients with AAV with sequential urine samples of active stage and remission. AAV, antineutrophil cytoplasmic antibody-associated vasculitis; EGF, epidermal growth factor; uEGF, urinary EGF.

    View this table:
    Table 2

    General data of patients with AAV for measuring urinary EGF

    Of the 60 patients with AAV with sequential urine samples of both active stage and remission, the level of uEGF/Cr was significantly higher in remission than those in the active stage (2.75±1.03 vs 2.08±0.98, p<0.001, by linear mixed model). After adjusting for eGFR value, the level of uEGF/Cr in patients in remission was still significantly higher than that in the active stage (mean (95% CI): 2.62 (2.40 to 2.84) vs 2.21 (2.00 to 2.43), p=0.008). Forty-two out of 60 patients had an increase (group A), while 18 patients had a decrease (group B), in the level of uEGF/Cr in remission as compared with those in active stage (figure 2B). The proportion of patients with IFTA grade ≥2 in group B was significantly higher than that in group A (absolute frequencies and percentages: 14/18 (77.8%) vs 20/42 (47.6%), p=0.031, by χ2 test). As expected, the eGFR slope in group B was significantly lower than that of patients in group A (−3.59±5.83 vs 0.94±6.47 mL/min/1.73 m2 per year, p=0.014, by Student’s t-test) during follow-up.

    Association of uEGF/Cr levels with clinical–pathological parameters

    Next, we assessed the association between the uEGF/Cr levels and clinical–pathological parameters using simple linear regression. For both active stage and remission stage of AAV, uEGF/Cr was significantly associated with eGFR(log2) at sampling (β=0.60, p<0.001; β=0.74, p<0.001, respectively). The level of uEGF/Cr showed a negative association with IFTA grade (β=−0.37, p=0.005). However, there was no significant association between uEGF/Cr and CRP or BVAS. Note that analyses were based on log2-transformed uEGF/Cr value, untransformed value was also used for data analysis, and results were similar.

    Using the definition of renal response to treatment, treatment failure occurred in 32 out of 173 (18.5%) patients with active AAV. They experienced fast deterioration in renal function during the induction therapy, with a rapid rate of eGFR decline of −3.26±2.41 mL/min/1.73 m2 per month. Eight out of 32 patients progressed to irreversible ESRD within the first month despite early institution of immunosuppressive therapy. Among these 32 patients, their extrarenal manifestations were ameliorated at various degrees after treatment. Patients who were ‘treatment failure’ had significantly lower level of uEGF/Cr (samples collected prior to treatment) than patients who had renal response to induction therapy (1.65±1.22 vs 2.16±1.26, p=0.04, by Student’s t-test).

    uEGF/Cr and renal outcomes of AAV

    To determine whether uEGF/Cr could predict the loss of kidney function in patients with AAV after they achieved remission by the induction therapy, we analysed the association between uEGF/Cr level in remission stage and the slope of eGFR. Three or more eGFR values during follow-up were available for 133 out of 143 patients with AAV in remission. The level of uEGF/Cr was significantly associated with the eGFR slope (β=0.63, p<0.001, by linear regression).

    Moreover, among all the 173 patients with active AAV, 143 patients had follow-up data. We investigated the association between uEGF/Cr level at baseline (acute stage of AAV) and the composite endpoint, defined as ESRD or 30% reduction of eGFR. During the follow-up (median 18.0 months, IQR 6.0–39.0 months), 38 patients developed ESRD, and 59 patients reached the composite endpoint. Univariable Cox regression analysis showed that urinary protein at renal biopsy, eGFR, protocols of induction therapy and uEGF/Cr were significantly associated with the composite endpoint. Multivariable Cox regression analysis showed that the level of uEGF/Cr remained an independent predictor for the composite endpoint (HR 0.61, 95% CI 0.45 to 0.83, p=0.001), even after adjusting for age, gender, eGFR, urinary protein at renal biopsy, type of ANCA and treatment protocols (table 3). Time-dependent ROC analysis showed that the area under the ROC curve for the reciprocal of uEGF/Cr at 72 months was 0.66. At the cut-off value of 0.46 for the reciprocal of uEGF/Cr, the sensitivity was 63% and the specificity was 63%.

    View this table:
    Table 3

    Cox proportional hazard models for the composite endpoint in patients with AAV

    In order to evaluate the additive effect of uEGF/Cr on traditional clinical markers for renal progression of AAV, we applied nested Cox proportional hazards models for this analysis. In comparison with the base model (M0, including age and gender), adding traditional clinical parameters to the base model improved the fitting capacity. Addition of uEGF/Cr to these models further improved the model fit significantly (table 4).

    View this table:
    Table 4

    Comparison of nested models of survival analysis

    Discussion

    EGF is one of the typical peptide growth factors.27 28 In humans, the kidney is the main organ for EGF production.29 To be more specific, EGF expression can be found in the ascending portion of Henle’s loop and the distal tubule in the kidney.30 By activating the EGF receptors, EGF plays a variety of roles in controlling cell proliferation, differentiation, survival and motility.31–33 However, whether the level of uEGF is associated with the disease severity and renal outcome of AAV is still far from clear.

    In this study, similar to what has been reported in CKD cohorts, we observed a significant association between intrarenal EGF mRNA levels and eGFR in patients with AAV. Since uEGF is highly correlated with EGF mRNA levels,12 it is not surprising that the level of uEGF/Cr was correlated with eGFR. The association between EGF and the degree of IFTA in the renal histology confirmed previous reports in patients with CKD and primary glomerulonephritis.12 25 Meanwhile, we found that the uEGF/Cr level was associated with renal response to treatment. More importantly, the level of uEGF/Cr in remission stage was associated with the eGFR slope and the level of uEGF/Cr at baseline was an independent predictor for renal outcomes of AAV. While uEGF/Cr in the current study had moderately good sensitivity and specificity for predicting poor renal outcome, we found that with the addition of uEGF/Cr onto the traditional predictive model for AAV progression, the model’s predictive performance improved. As urinary EGF is considered to reflect the degree of functional distal tubular cell mass, the reduced level of uEGF could be a reflection of the degree of tubular damage present in a patient with AAV. This could be the consequence of severity of ongoing autoimmune disease or a consequence of past immune damage to the tubular compartment. Both scenarios could explain the significant association between the lower level of uEGF/Cr and worse long-term outcome.

    EGF is a main trophic factor for tubular cells and modulates tissue response to injury.30 Moreover, exogenous EGF enhanced tubular cell repair processes, suggesting that EGF may regulate tubular recovery to damage.34 Regarding the underlying protection mechanism of EGF in kidneys, studies have shown that high EGF levels reduced renal epithelial sodium channel activity, which could alleviate glomerular and renal tubular damage.35 36 However, the protective mechanism of EGF in AAV progression needs to be further explored.

    Strengths of the study include the assessment of association of uEGF/Cr level with active and remission AAV and the sequential longitudinal sampling from patients to evaluate the association of uEGF/Cr levels with renal progression of AAV. Limitations include the single-centre design with modest sample size, and relatively short and varied duration of follow-up, necessitating a validation in larger, independent multiethnic AAV study cohort. Important questions for future studies are whether uEGF level could predict treatment response and whether patients in remission with lower uEGF/Cr profiles should be monitored more closely for disease recurrence and potentially more aggressive therapy.

    In conclusion, we show that lower uEGF/Cr level is associated with more severe renal disease, renal resistance to treatment and higher risk of progression to composite endpoint in patients with AAV. uEGF/Cr level may be a useful non-invasive biomarker to assess the degree of tubular damage and the potential for the transition into chronic progressive kidney disease in patients with AAV, which warrants validation in an independent, larger and multicentre cohort study.

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    View Abstract

    Footnotes

    • Handling editor Josef S Smolen

    • Contributors LW and X-QL participated in urine sample and clinical information collection. LW participated in the measurement of ELISA. LW, TG, SE and W-JJ participated in data analysis and interpretation. LW wrote the manuscript. MC, W-JJ, LW, TG and SE revised the manuscript. MC, W-JJ, MK and M-HZ participated in the study design and approved the final manuscript. All authors reviewed and approved the manuscript’s content before submission.

    • Funding This study was supported by grants from the National Key Research and Development Program (no. 2016YFC0906102), National Natural Science Fund (nos. 81425008 and 81621092), Peking University Health Science Center (no. BMU2017CJ002), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (P30DK081943 to MK) and from the University of Michigan Health System and Peking University Health Sciences Center Joint Institute for Translational and Clinical Research (no. BMU2017JI005) and from the National Institute of Health.

    • Competing interests None declared.

    • Patient consent Obtained.

    • Ethics approval The research was approved by the ethics committees of Peking University First Hospital and the ethics committees of the University of Michigan.

    • Provenance and peer review Not commissioned; externally peer reviewed.