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Keywords:

  • biomarker;
  • complement;
  • IgA nephropathy;
  • MBL;
  • prognosis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

There is accumulating evidence to support a hypothesis of the activation of the lectin complement pathway in immunoglobulin A nephropathy (IgAN). The glomerular deposition of mannose-binding lectin (MBL), an initiator of the lectin pathway, has been identified, but its clinical significance has not been defined consistently. The aim of the present study was to investigate the value of glomerular MBL deposition as a useful histological biomarker in evaluating the severity and predicting the prognosis of IgAN. We included all consecutive patients with biopsy-proven primary IgAN from December 2008 to July 2010. Renal deposition of MBL was detected by immunofluorescence. The biopsy material from 131 patients (72 men) was thus used for MBL staining. The deposition of MBL was observed in a predominantly mesangial pattern in 45 patients (34·35%), which presented as global or segmental deposition. Compared with the patients without glomerular MBL deposition, those with glomerular MBL deposition had more severe proteinuria, decreased renal function, lower levels of serum albumin and a greater possibility of hypertension at the time of renal biopsy; they had more severe histological changes according to the Oxford classification (i.e. mesangial hypercellularity, segmental glomerulosclerosis, endocapillary hypercellularity and tubular atrophy/interstitial fibrosis), and their ratio presented an increase as the histopathological phenotypes segregated according to Lee's classification; furthermore, the follow-up data demonstrated that they had a lower renal remission rate. In conclusion, glomerular MBL deposition may predict a poor prognosis, and thus can be a new prognostic factor in IgA nephropathy.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis worldwide, comprising 20–45% of all primary glomerular diseases [1, 2]. Currently, it is well recognized that 30–40% of patients with IgAN reach end-stage renal disease (ESRD) within 20 years from apparent disease onset, underscoring that IgAN is not a benign condition [3, 4]. The clinical spectrum of IgAN is highly variable, ranging from minor urinary abnormalities to rapidly progressive renal failure. The variability in clinical course makes it necessary to investigate clinical and histological predictors for the progression of IgAN.

The major pathohistological characteristics of IgAN are mesangial cell proliferation and matrix expansion with granular deposition of IgA, predominantly polymeric immunoglobulin (Ig)A1, and complement (C) 3 in the glomerular mesangial areas. Recently it has been demonstrated that the glomerular deposits containing aberrantly glycosylated IgA1 trigger the local production of cytokines and growth factors, leading to mesangial cell activation and local complement activation [5-8]. The complement system can be activated via three pathways: the classical, alternative and lectin pathways. Substantial in-vivo and in-vitro evidence has recognized that the alternative pathway is involved in the pathogenesis of IgAN, whereas the classical pathway is dissociated from IgAN [9]. Recently, there is accumulating evidence to support a hypothesis of the occurrence of the lectin pathway activation in IgAN [10-13]. Mannose-binding lectin (MBL), a member of the collectin family of proteins with a C-terminal lectin domain and a collagenous backbone [14], initiates the lectin pathway by reacting with the MBL-associated serine proteases MASP-1 and MASP-2. The glomerular deposition of MBL has been identified, but its clinical significance has not been defined consistently [13, 15, 16].

In the present cohort study, to investigate the value of glomerular MBL deposition as a useful histological biomarker in evaluating the severity and predicting the prognosis of IgAN, we included 131 patients with IgAN and performed a mean follow-up of 39·8 months. We detected the renal deposition of MBL with immunofluorescence and analysed the relationship between glomerular MBL deposition and clinical and histological features, which are generally acknowledged to be independent predictors of progressive renal disease or renal failure [17-19]. We also evaluated the difference of renal remission rates between the patients with and without MBL deposition. To the best of our knowledge, the present study is the first to evaluate the association between renal remission rates and glomerular MBL deposition with the follow-up data, and includes the largest number of IgAN patients on this issue.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

Subjects

The present study enrolled consecutively patients who underwent renal biopsy in our department and who were diagnosed as primary IgAN from December 2008 to July 2010. A signed informed consent was obtained before renal biopsy. This study was performed according to recommendations outlined in the Declaration of Helsinki Principles (IV Adaptation) and approved by the Ethics Committee and the Research Board of our institution. The day of renal biopsy was considered as the start day of the study. No patient was administered corticosteroids and immunosuppressants before the start of the present study.

The clinical parameters of all the included patients were obtained before renal biopsy, such as routine urinalysis, serum albumin and creatinine (SCr) and 24-h urinary protein excretion. The estimated glomerular filtration rate (eGFR) was calculated using the four-variable Modification of Diet in Renal Disease (MDRD) formula [20], and the result was reduced by 25·8% for women. Hypertension was said to be present if the arterial blood pressure was at or above 140/90 mmHg, or if levels less than 140/90 mmHg were reached with anti-hypertensive medications.

Evaluation of renal histopathological lesion

The diagnosis of IgAN was based on histological assessment of renal tissue with haematoxylin and eosin, Masson's trichrome, periodic acid-Schiff and methenamine silver for light microscopy and staining with IgG, IgA, IgM, C3 and C1q for immunofluorescence. Two experienced pathologists who were blinded to the patients' data and results from the other observer evaluated the renal tissue separately. Differences between the two pathologists were resolved by re-reviewing the sections and coming to a consensus.

The severity of renal histological lesions was evaluated according to Lee's classification [21] and the Oxford classification [22]. First, we divided the included patients into three groups according to Lee's classifications; those fulfilling the histopathological criteria of Lee-I or -II were defined as mild renal lesions (group 1), those with Lee-III as moderate renal lesions (group 2) and those with Lee-IV or -V as severe renal lesions (group 3). We then further evaluated four variables of renal histopathological lesion recommended by the Oxford classification; that is, mesangial proliferation, segmental glomerulosclerosis, endocapillary hypercellularity and tubular atrophy/interstitial fibrosis, which have independent value in predicting renal outcome.

Treatment protocol

As conducted in previously described methods [10], the patients with haematuria, proteinuria of less than 1 g/24 h and normal renal function, considered to be at low risk for loss of kidney function, received non-immunosuppressive therapy (e.g. angiotensin converting enzyme inhibitors (ACEI) and/or angiotensin II receptor blockers (ARB), fish oil, statins and anti-platelets), whereas those with proteinuria of 1·0 g/24 h or more and active lesions in renal biopsies, identified as at high risk for loss of kidney function, were treated with immunosuppressive therapy [23-26]; that is, corticosteroids alone or combined with other immunosuppressive drugs (e.g. cyclophosphamide, cyclosporin A and mycophenolate mofetil). If necessary, the latter could also be administered with non-immunosuppressive therapy.

Outcome definitions

Patient medical records were reviewed in December 2012. The primary end-point of the study was renal remission defined as stable or improved renal function (i.e. no ‘progressive renal impairment’) with reduction of proteinuria by >50%, proteinuria less than 3 g/24 h and serum albumin >30 g/l. Progressive renal impairment was defined as a reduction in eGFR ≥ 5 ml/min/1·73 m2/year or received renal replacement therapy during the follow-up period.

Detection of renal deposition of MBL by immunofluorescence

For immunofluorescence staining, unfixed renal tissue was embedded in optical cutting temperature (OCT) compound (Sakura Tissue-tek, Bayer, Torrance, California, USA), snap-frozen in a mixture of isopentane and dry ice and stored at −80°C. Subsequently, 4-μm sections were placed on slides and stored at −20°C until immunostaining. The slides were dried before use, and then fixed in acetone at 4°C for 10 min. The slides were washed three times in phosphate-buffered saline (PBS). The blocking buffer was applied to the renal tissue slides for 30 min at room temperature. To explore the location of glomerular MBL deposition, we performed immunofluorescent double staining of MBL and fibronectin, which is a mesangial cell marker. The mixture of mouse anti-human MBL monoclonal antibodies (Abcam, Cambridge, MA, USA) diluted 1:40 in PBS and rabbit anti-human fibronectin polyclonal antibodies (Dako, Glostrup, Denmark) diluted 1:500 in PBS was applied to the slides and incubated overnight at 4°C as primary antibodies. The slides were washed three times in PBS on the second day. A mixture of Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies diluted 1:200 in PBS and Alexa Fluor 594-conjugated goat anti-rabbit IgG antibodies diluted 1:200 in PBS was used as secondary antibodies for 30 min at 37°C. The slides were washed in PBS three times and coverslipped using glycerol-Tris (hydroxymethyl) aminomethane (Tris) mounting medium. Renal deposition of MBL and fibronectin by immunofluorescence was detected using a Nikon A1 confocal microscope.

Statistical analysis

The normally distributed data (e.g. age) were expressed as mean ± standard deviation, the skewed data (e.g. SCr, eGFR, proteinuria, serum albumin, C3, C4 and IgA) were expressed as median and range and the categorical data were expressed as absolute frequencies and percentages. Differences were compared for normally distributed data with the independent t-test, differences for skewed distributed data with the Mann–Whitney U-test and differences for categorical data with the χ2 test. Spearman's correlation was applied for analysing correlations. A two-tailed P-value ≤ 0·05 was considered statistically significant. All statistical analyses were performed with spss version 16·0 (SPSS, Inc., Chicago, IL, USA).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

Characteristics of IgAN patients

One hundred and sixty-two patients were diagnosed with IgAN by renal biopsy performed in our department between December 2008 and July 2010. MBL immunofluorescence staining was not performed on 31 patients who had insufficient renal tissue (fewer than two glomeruli). The biopsy material from the remaining 131 patients (72 men) was thus used for MBL staining.

MBL immunofluorescence staining was observed in 45 patients (34·35%) which presented as global or segmental deposition (Fig. 1), both of which were regarded as MBL-positive. MBL was co-localized with fibronectin, which indicated that the glomerular deposition of MBL presented predominantly in a mesangial pattern. The renal tissue of the remaining 86 patients stained negative for MBL.

figure

Figure 1. Renal tissue from immunoglobulin A nephropathy (IgAN) patients was stained for the presence and localization of mannose-binding lectin (MBL), which showed that MBL was co-localized with fibronectin, a mesangial cell marker. Representative images are shown. (a–d) Global deposition and co-localization of MBL with fibronectin: (a) global deposition of MBL, (b) deposition of fibronectin, (c) merging of MBL and fibronectin, (d) negative control with only secondary antibody at the same dilution; (e–h) segmental deposition and co-localization of MBL with fibronectin: (e) segmental deposition of MBL, (f) deposition of fibronectin, (g) merging of MBL and fibronectin, (h) negative control with only secondary antibody at the same dilution. Magnification ×400.

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The clinical characteristics of IgAN patients among the different groups are described in Table 1. There were no significant differences in mean age, sex and rate of loss to follow-up among the three groups. The ratio of patients with hypertension and the levels of urinary protein excretion showed no significant differences between groups 1 and 2, but they were significantly higher in group 3 than those in groups 1 and 2. The levels of SCr presented a significant increase as the histopathological phenotypes aggravated, whereas the levels of eGFR showed an opposite trend.

Table 1. Clinical characteristics of immunoglobulin A nephropathy (IgAN) patients
 Group 1 (n = 41)Group 2 (n = 46)Group 3 (n = 44)P-value
1 versus 21 versus 32 versus 3
  1. eGFR: estimated glomerular filtration rate; IgAN: immunoglobulin A nephropathy; SCr: serum creatinine; s.d.: standard deviation.

Mean age (years) (mean ± s.d.)32·02 ± 12·1835·93 ± 10·8736·80 ± 11·790·3180·1970·978
Male/female23/1821/2528/160·3930·5130·096
Hypertension3/415/4631/440·717<0·001<0·001
SCr [μmol/l, median (range)]66 (38–122)77·5 (33–212)149 (51–932)0·001<0·001<0·001
eGFR [ml/min/1·73 m2, median (range)]123·47 (82·58–176·80)96·21 (40·94–166·56)57·49 (8·92–121·48)<0·001<0·001<0·001
Proteinuria [g/24 h, median (range)]1·12 (0·10–3·41)1·08 (0·08–7·25)3·00 (0·41–8·13)0·899<0·001<0·001
Loss to follow-up4/415/467/441·0000·5230·546

The histopathological parameters are shown in Table 2. The patients' ratios of M0:M1, S0:S1, E0:E1 and T0:T1:T2 decreased gradually with the aggravation of histopathological phenotypes.

Table 2. Histopathological characteristics of immunoglobulin A nephropathy (IgAN) patients
 Group 1 (n = 41)Group 2 (n = 46)Group 3 (n = 44)P-value
1 versus 21 versus 32 versus 3
  1. According to the Oxford classification, four variables were evaluated; that is, mesangial proliferation (M), segmental glomerulosclerosis (S), endocapillary hypercellularity (E) and tubular atrophy/interstitial fibrosis (T). IgAN: immunoglobulin A nephropathy.

M0/M140/136/1023/210·008<0·0010·014
S0/S133/815/313/41<0·001<0·0010·003
E0/E137/429/1717/270·005<0·0010·034
T0/T1/T240/1/025/19/22/17/25<0·001<0·001<0·001

Correlations between glomerular MBL deposition and clinical parameters

There were no significant differences between the patients with and without glomerular MBL deposition in sex distribution and age at the time of renal biopsy (P = 0·209, 0·713).

Compared with the patients without MBL deposition, those with glomerular MBL deposition had significantly higher levels of SCr (P < 0·001) (Fig. 2a), urinary protein excretion (P = 0·001) (Fig. 2b) and ratio of hypertension (P < 0·001) (Fig. 2c), but significantly lower levels of eGFR (P < 0·001) (Fig. 2d) and serum albumin (P = 0·002) (Fig. 2e).

figure

Figure 2. Comparison of the clinical parameters between the patients with and without glomerular mannose-binding lectin (MBL) deposition [Mann–Whitney U-test for serum albumin and creatinine (SCr), urinary protein excretion (UPE), estimated glomerular filtration rate (eGFR) and serum albumin and χ2 test for hypertension]. (a) SCr; (b) UPE; (c) hypertension; (d) eGFR; (e) serum albumin. The horizontal solid lines indicate the median and range values for each group.

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No significant differences were detected in the levels of serum C4, C3 and IgA and haematuria [expressed by red blood cell (RBC) counts per high-power field] between the two groups of patients (P = 0·068, 0·561, 0·716, 0·078).

Correlations between glomerular MBL deposition and histopathological parameters

First, the ratio of the patients with glomerular MBL deposition presented a gradual increase as the histopathological phenotypes segregated according to Lee's classification (Table 1, Fig. 3a). It should be noted that the difference between groups 1 and 2 was statistically significant (P = 0·047), whereas the difference between groups 2 and 3 was not (P = 0·136).

figure

Figure 3. Comparison of the histological parameters between the patients with and without glomerular mannose-binding lectin (MBL) deposition with the χ2 test. (a) Comparison of the proportions of patients with and without glomerular MBL deposition among different renal histopathological phenotypes according to Lee's classification; (b–e) comparison of the four histological parameters according to the Oxford classification; (b) mesangial hypercellularity (M); (c) segmental glomerulosclerosis (S); (d) endocapillary hypercellularity (E); (e) tubular atrophy/interstitial fibrosis (T).

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In addition, the patients with MBL deposition had more severe histopathological features according to the Oxford classification; that is, more M1, S1, E1 and T1/T2 than those without MBL deposition (P < 0·001, <0·001, <0·001, 0·001) (Table 2, Fig. 3b–e)

Correlation between glomerular MBL deposition and renal remission

In the present cohort study, 16 of 131 included patients were lost to follow-up. Thus, the medical records of the remaining 115 patients were reviewed in December 2012. The mean follow-up time was 39·8 ± 5·7 months.

In all the 115 IgAN patients, those with glomerular MBL deposition had a significantly lower ratio of renal remission than those without MBL deposition (P < 0·001) (Fig. 4a). Considering different risk stratifications for loss of kidney function and the effect of different treatment protocols on disease progression, the patients were stratified into a low-risk non-immunosuppressive therapy group and a high risk immunosuppressive therapy group. In both subgroups, the patients with MBL deposition had significantly lower ratios of renal remission (P < 0·001, <0·001) (Fig. 4b,c).

figure

Figure 4. Comparison of the proportions of renal remission between the patients with and without glomerular mannose-binding lectin (MBL) deposition with the χ2 test. (a) Comparison in all patients who completed the whole period of follow-up (n = 115); (b) comparison in patients with non-immunosuppressive therapy (n = 43); (c) comparison in patients with immunosuppressive therapy (n = 72).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

In recent years, there has been increasing evidence that the lectin pathway is involved in IgAN, but the clinical significance of glomerular MBL deposition as a useful histological biomarker is still undetermined. Endo et al. reported that the IgAN patients with MBL/MASP-1 deposition had younger ages and shorter durations of the disease prior to renal biopsy than those without MBL/MASP-1 deposition, and had a more distinct activity index than chronicity index [15]. The study by Roos et al., with 60 IgAN patients, showed that glomerular staining for MBL is associated with more severe proteinuria, renal dysfunction and histological parameters (i.e. mesangial proliferation, extracapillary proliferation, global sclerosis and interstitial infiltration) [13]. Conversely, Hisano et al. showed that there no differences in clinical and pathological severity parameters for patients with mesangial MBL deposition [16]. These previous studies gave inconsistent results.

In the present cohort study, we included 131 IgAN patients and performed a mean follow-up of 39·8 months. MBL immunofluorescence staining was observed in 45 patients (34·35%). MBL was co-localized with fibronectin, indicating that the glomerular deposition of MBL presented predominantly in a mesangial pattern. We then correlated glomerular MBL deposition with generally acknowledged clinical prognostic factors for the development of ESRD; that is, renal function (expressed by SCr and eGFR), proteinuria, arterial hypertension and serum albumin at the time of renal biopsy [17-19]. We also evaluated the relationship between MBL deposition and histological parameters. In terms of histological analysis, we grouped together the patients with Lee's classification and evaluated the four parameters (i.e. mesangial hypercellularity, segmental glomerulosclerosis, endocapillary hypercellularity and tubular atrophy/interstitial fibrosis) which have independent value in predicting renal outcome of IgAN according to the Oxford classification. The selection of generally acknowledged histological parameters made our conclusion more reliable. More importantly, we investigated the association between glomerular MBL deposition and renal remission according to the follow-up data. According to the present study, the IgAN patients with glomerular MBL deposition had more severe proteinuria, decreased renal function, lower levels of serum albumin and a greater possibility of hypertension at the time of renal biopsy than those without MBL deposition; they had more severe histological changes (i.e. more severe mesangial hypercellularity, segmental glomerulosclerosis, endocapillary hypercellularity and tubular atrophy/interstitial fibrosis), and their ratio presented an increase as the histopathological phenotypes aggravated according to Lee's classification; according to the follow-up data, they had a lower renal remission rate. Therefore, we concluded that glomerular MBL deposition may be a new prognostic predictor in IgA nephropathy.

We have reported previously that urinary MBL was elevated with aggravation of renal lesions [10]. In this cohort of IgAN patients, the patients with glomerular MBL deposition had higher levels of urinary MBL than those without MBL deposition (P < 0·001). This may support further the hypothesis that elevated urinary MBL may derive from MBL accumulation in renal tissue instead of circulation. In terms of the cause of MBL deposition, we hypothesized that MBL can recognize the protease-damaged surface of glomerular resident cells or apoptotic cells in IgAN, and thus MBL and/or the immune complexes containing MBL accumulate in renal tissue.

However, the present study has some limitations: as an observational study, the relationship between glomerular MBL staining and progression of IgAN should be interpreted cautiously in terms of association instead of causality. Furthermore, its external validity might be limited as a single-centre study.

In summary, we conclude that the IgAN patients with glomerular MBL deposition had more severe clinical and histological features and a significantly lower renal remission rate. Glomerular MBL deposition might be an important histological predictor for the prognosis of IgA nephropathy.

Disclosure

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References

The authors declare no competing financial interests.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosure
  8. References