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Implication of Urinary Complement Factor H in the Progression of Immunoglobulin A Nephropathy

  • Maojing Liu,

    Affiliations Renal Division, Department of Medicine, Peking University First Hospital; Beijing 100034, China, Institute of Nephrology, Peking University; Beijing 100034, China, Key Laboratory of Renal Disease, Ministry of Health of China; Beijing 100034, China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education; Beijing 100034, China

  • Yuqing Chen ,

    cyq@bjmu.edu.cn

    Affiliations Renal Division, Department of Medicine, Peking University First Hospital; Beijing 100034, China, Institute of Nephrology, Peking University; Beijing 100034, China, Key Laboratory of Renal Disease, Ministry of Health of China; Beijing 100034, China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education; Beijing 100034, China

  • Jingjing Zhou,

    Affiliations Renal Division, Department of Medicine, Peking University First Hospital; Beijing 100034, China, Institute of Nephrology, Peking University; Beijing 100034, China, Key Laboratory of Renal Disease, Ministry of Health of China; Beijing 100034, China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education; Beijing 100034, China

  • Ying Liu,

    Affiliations Renal Division, Department of Medicine, Peking University First Hospital; Beijing 100034, China, Institute of Nephrology, Peking University; Beijing 100034, China, Key Laboratory of Renal Disease, Ministry of Health of China; Beijing 100034, China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education; Beijing 100034, China

  • Fengmei Wang,

    Affiliations Renal Division, Department of Medicine, Peking University First Hospital; Beijing 100034, China, Institute of Nephrology, Peking University; Beijing 100034, China, Key Laboratory of Renal Disease, Ministry of Health of China; Beijing 100034, China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education; Beijing 100034, China

  • Sufang Shi,

    Affiliations Renal Division, Department of Medicine, Peking University First Hospital; Beijing 100034, China, Institute of Nephrology, Peking University; Beijing 100034, China, Key Laboratory of Renal Disease, Ministry of Health of China; Beijing 100034, China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education; Beijing 100034, China

  • Yanfeng Zhao,

    Affiliations Renal Division, Department of Medicine, Peking University First Hospital; Beijing 100034, China, Institute of Nephrology, Peking University; Beijing 100034, China, Key Laboratory of Renal Disease, Ministry of Health of China; Beijing 100034, China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education; Beijing 100034, China

  • Suxia Wang,

    Affiliation Laboratory of Electron Microscopy, Peking University First Hospital, Peking University First Hospital; Beijing 100034, China

  • Lijun Liu,

    Affiliations Renal Division, Department of Medicine, Peking University First Hospital; Beijing 100034, China, Institute of Nephrology, Peking University; Beijing 100034, China, Key Laboratory of Renal Disease, Ministry of Health of China; Beijing 100034, China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education; Beijing 100034, China

  • Jicheng Lv,

    Affiliations Renal Division, Department of Medicine, Peking University First Hospital; Beijing 100034, China, Institute of Nephrology, Peking University; Beijing 100034, China, Key Laboratory of Renal Disease, Ministry of Health of China; Beijing 100034, China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education; Beijing 100034, China

  • Hong Zhang,

    Affiliations Renal Division, Department of Medicine, Peking University First Hospital; Beijing 100034, China, Institute of Nephrology, Peking University; Beijing 100034, China, Key Laboratory of Renal Disease, Ministry of Health of China; Beijing 100034, China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education; Beijing 100034, China

  • Minghui Zhao

    Affiliations Renal Division, Department of Medicine, Peking University First Hospital; Beijing 100034, China, Institute of Nephrology, Peking University; Beijing 100034, China, Key Laboratory of Renal Disease, Ministry of Health of China; Beijing 100034, China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education; Beijing 100034, China

Implication of Urinary Complement Factor H in the Progression of Immunoglobulin A Nephropathy

  • Maojing Liu, 
  • Yuqing Chen, 
  • Jingjing Zhou, 
  • Ying Liu, 
  • Fengmei Wang, 
  • Sufang Shi, 
  • Yanfeng Zhao, 
  • Suxia Wang, 
  • Lijun Liu, 
  • Jicheng Lv
PLOS
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Abstract

Background

After activation, the complement system is involved in the pathogenesis of Immunoglobulin A nephropathy (IgAN). Complement factor H (CFH) is a crucial inhibitory factor of the alternative pathway of the complement system. The study investigated the effects of urinary CFH levels on IgAN progression.

Methods

A total of 351patients with IgAN participated in this study. They were followed up for an average of 51.8±26.6 months. Renal outcome was defined as a composite endpoint, that included instances of end-stage renal disease (ESRD),≥ 50% decline in estimated glomerular filtration rate (eGFR) or doubling of plasma creatinine levels. Urinary CFH levels were measured by enzyme-linked immunosorbent assay and calculated as the ratio of urinary CFH over creatinine (uCFH/uCr).

Results

In the whole cohort, uCFH/uCr values were associated with disease progression either as continuous [log(uCFH/uCr)] or categorical traits (dichotomous and quartile variables) after adjusting for eGFR, proteinuria, mean arterial blood pressure, histological grading and immunosuppressive therapy in the Cox proportional hazard model. Kaplan-Meier analysis showed that higher uCFH/uCr values at baseline predicted worse renal outcome during follow-up (log-rank, P<0.001). Receiver operating characteristic curve (ROC) analysis showed that log(uCFH/uCr) had predictive value for renal outcome (area under curve [AUC]=0.745), and the AUC increased to 0.805 after being incorporated into baseline eGFR and proteinuria. In subgroup analysis with eGFR≥60 mL/min/1.73m2, log(uCFH/uCr) had better predictive value (AUC= 0.724, P=0.002) for renal outcome compared to eGFR (AUC = 0.582, P=0.259) and proteinuria (AUC = 0.615, P=0.114).

Conclusions

Urinary CFH levels are associated with renal function decline and increased urinary CFH levels are a risk factor for progression of IgA nephropathy.

Introduction

Complement factor H (CFH) is one of the crucial inhibitory factors of the alternative pathway of the complement system, acting to accelerate inactivation of C3b [1].Studies suggest that activation of the complement alternative pathway may be involved in Immunoglobulin A nephropathy (IgAN) pathogenesis [2,3,4,5,6,7,8,9,10,11]. Our previous study and results from other studies demonstrated deposition of CFH and C3b in renal tissue of patients with IgAN, and higher levels of serum/urinary CFH were associated with disease activity [12,13,14,15]. However, it has remained unknown whether urinary CFH levels are associated with disease progression.

In this study, we measured urinary CFH levels in our IgAN cohort, analyzed the correlation between these levels and clinical/histological parameters, and evaluated the value of urinary CFH levels for predicting renal outcome.

Materials and Methods

Subjects

This study was approved by Clinical Research Ethics Committee of Peking University First Hospital. All individuals included in this study signed consent forms so that their information could be stored in the hospital database and used for research.

A total of 351patients with IgAN were included in this study. Patients who had participated in a previous CFH study were not included [15].Clinical parameters, including age, gender, blood pressure, proteinuria, plasma creatinine levels and other physical and biochemical characteristics were collected on the day of renal biopsy and during follow-up. The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation [16]. Renal pathological lesions were graded according to the Hass classification system [17]. Histological grades were divided into groups A (Hass I, II, and III) and B (Hass IV and V) [18] (Table 1).

Each patient was followed up at least for 12 months. The renal composite endpoint was defined as: 1) end-stage renal disease (ESRD) (eGFR<15mL/min/ 1.73 m2); 2) eGFR decline ≥50% from the baseline value; 3) doubling of plasma creatinine levels.

Measurement of urinary CFH by Enzyme-linked Immunosorbent Assay (ELISA)

Urine spots were collected on the day of renal biopsy and stored at -80°C until measurement. Urinary CFH concentrations were measured by ELISA described previously, with minor modifications [15]. Briefly, plates (Nalge-Nunc, Rochester, NY) were coated with goat anti-human factor H (Calbiochem, Darmstadt, Germany) at a concentration of 10μg/mL in 0.05 M bicarbonate buffer (pH = 9.6). Plates coated with bicarbonate buffer alone were also prepared simultaneously as antigen-free controls. The reaction volume was 100 μL. Urine samples from patients with IgAN and healthy controls were added to wells in duplicate. After incubation and washing, 1:1000 diluted mouse anti-human CFH (US Biological, Swampscott, MA, USA) was added. Alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO, USA) was added as a second antibody following three washes. The absorbance was measured at 405 nm with an EL312 Bio-Kinetics microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Commercially available highly purified human factor H was used to establish the standard curve.

The urinary CFH measurements were corrected by urinary creatinine concentration, and the ratios of urinary CFH over urinary creatinine (uCFH/uCr) were used for analysis.

Detection of CFH deposition by immunofluorescence staining

Renal tissue samples were obtained from IgAN renal biopsy specimens. Paraffin-embedded blocks were cut into sections of 4-μm-thick sections and mounted on pre-coated slides. The paraffin section was deparaffinized with xylene and dehydrated with ethanol. Endogenous peroxidase activity was blocked by incubating sections in a 3% H2O2 solution at room temperature for 30 min. Antigen retrieval was performed by incubating the sections in pepsin at 37°C for 1h. After blocking with phosphate buffered saline (PBS) containing 3% bovine serum albumin for 30min, the sections were incubated overnight at 4°C with primary goat anti-human CFH antibody (1:8000, diluted in PBS). Bound antibodies were detected with biotinylated rabbit anti-goat IgG. Diaminobenzidine was used as a chromogen.

Statistical analyses

Baseline features were reported as mean±SD, median (inter-quartile range [IQR]) for continuous variables, and ratios/percentages for categorical data. One-way post-hoc ANOVA using Bonferroni multiple comparison, Chi-square test, Mann-Whitney U test, and Kruskal-Wallis test was used to test for differences between groups. Spearman correlation coefficient was used to determine the correlation between urinary CFH levels and clinical variables.

A Cox proportional hazards model was used to analyze the association between urinary CFH levels and renal outcome in the whole group, as well as in subgroups with eGFR≥60 and eGFR<60mL/min/1.73m2. Both log (uCFH/uCr) and uCFH/uCrby quartiles and median were analyzed in the Cox models. The lowest group in quartiles or median was defined as the reference group in each analysis. Hazard ratio was tested in unadjusted and three adjusted Cox models. Survival curves were derived from the Kaplan—Meier analysis and compared by log-rank test. Receiver operating characteristic (ROC) analysis was used to assess the predictive power of log(uCFH/uCr).

Proportional hazards assumptions were verified by testing the interaction of survival time and log (uCFH/uCr), uCFH/uCr quartiles, and (low vs. high) uCFH/uCr (P = 0.269, P = 0.161, and P = 0.236, respectively). All statistical tests were performed using SPSS version 13.0.

Results

Urinary CFH levels at baseline correlated with clinical/histological parameters

Urinary CFH levels are shown as uCFH/uCr. The uCFH/uCr (34.5 ng/mg [0.0–9220.2]) at baseline was correlated with baseline eGFR (r = −0.347, P<0.001, Fig 1A), baseline proteinuria (r = 0.461, P<0.001, Fig 1B) and pathologic changes. After adjusting for baseline proteinuria, urinary CFH levels remained correlated with eGFR (r = −0.291, P<0.001). Patients with more diffuse and severe histological lesions (Hass IV and V) had higher urinary CFH levels compared to those with mild focal lesions (Hass I,II, and III) (P<0.001) (Fig 1C).

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Fig 1. Correlation between urinary CFH levels and clinical/histologic parameters.

A. Correlation between urinary CFH levels and baseline eGFR. Urinary CFH levels are shown as log(uCFH/uCr). Spearman correlation analysis was used to determine the correlation between urinary CFH levels and baseline eGFR. Spearman's r = −0.347, P<0.001, and after adjusting for baseline proteinuria r = −0.291, P<0.001. B. Correlation between urinary CFH levels and baseline proteinuria. Urinary CFH level are shown as log(uCFH/uCr). Spearman r-value and the P-value are shown. C. Urinary CFH levels and pathologic changes. Patients were divided into A (Hass I, II and III) and B (Hass IV and V) groups according to the Hass classification system. Urinary CFH levels were compared using the non-parametric Mann-Whitney U test.

https://doi.org/10.1371/journal.pone.0126812.g001

Immunofluorescence staining of CFH showed increased CFH deposition in the glomeruli of patients with IgAN compared to normal controls (S1 Fig).

Urinary CFH levels associated with renal survival in the whole cohort

Twenty-seven patients (7.7%) developed ESRD, 10 patients (2.8%) had a ≥50%eGFR decline, and 3 patients (0.85%) had doubled plasma creatinine levels during follow-up (51.8±26.6 months).

Patients were divided into four groups by quartiles of uCFH/uCr; patients in the first quartile had the lowest uCFH/uCr values(0.0–13.0ng/mg).Baseline data showed that patients in the third and fourth quartiles had more proteinuria, lower eGFR, diffuse histological lesions, and higher ESRD (Table 2).Univariate analyses revealed that clinical features, including uCFH/uCr, mean arterial pressure (MAP), eGFR, proteinuria, histological grading, and immunosuppressive therapy, were associated with renal outcome(S1 Table).We therefore performed unadjusted and adjusted models in further multivariate Cox’s regression analyses. Model I was adjusted for eGFR, proteinuria and MAP. Histological grading was added to Model II based on Model I, and immunosuppressive therapy was added to the model III based on model II. In all four Cox models, uCFH/uCr was associated with disease progression as continuous and categorical traits. When the lowest quartile uCFH/uCr was selected as a reference group, the risk of kidney function decline increased in the third and fourth quartile (Table 3). Kaplan-Meier curve analysis showed that higher uCFH/uCr ratios predicted worse renal outcome during follow-up (log rank, P< 0.001) (Fig 2A).

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Fig 2. Urinary CFH levels predicted renal survival in the IgAN cohort.

The survival curve was derived from Kaplan—Meier analysis and compared by log-rank test. A. Urinary CFH levels and renal survival in the whole cohort by quartiles. Patients were divided into four groups according to uCFH/uCr quartiles; the first quartiles had the lowest uCFH/uCr values (0.0–13.0ng/mg). Patients in the third and fourth quartiles had more frequently progressed to composite endpoints. B. Median urinary CFH levels and renal survival in the whole cohort. All individuals were divided into two groups by median uCFH/uCr values. The patients with higher uCFH/uCr had increased risk of progressing to the composite endpoint. C. Median urinary CFH levels and renal survival in the subgroup of patients with eGFR≥60mL/min/1.73m2. Individuals with eGFR≥60mL/min/1.73m2 were divided into two groups by median uCFH/uCr. The patients with higher uCFH/uCrhad increased risk of progress to a composite endpoint. D