Segmental Membranous Glomerulonephritis in Children - CiteSeerX

Segmental Membranous Glomerulonephritis in Children: Comparison with Global Membranous Glomerulonephritis Mina Obana,* Koichi Nakanishi,* Mayumi Sako,* Nahoko Yata,* Kandai Nozu,† Ryojiro Tanaka,‡ Kazumoto Iijima,§ and Norishige Yoshikawa* *Department of Pediatrics, Wakayama Medical University, Wakayama City, Wakayama, †Department of Pediatrics, Kobe University Graduate School of Medicine, and ‡Department of Nephrology, Hyogo Prefectural Kobe Children’s Hospital, Kobe, Hyogo, and §Department of Nephrology, National Center for Child Health and Development, Setagaya, Tokyo, Japan Generally, idiopathic membranous glomerulonephritis (MGN) is a global glomerular disease that affects the whole of the glomerulus. However, idiopathic segmental MGN (SMGN) that shows IgG deposits in a portion of the glomerulus is encountered often. For clarification of whether SMGN is the same entity as idiopathic global MGN (GMGN), the two diseases were compared. From 1978 to 2004, 38 children (11 with SMGN and 27 with GMGN) received a diagnosis of idiopathic MGN. Immunofluorescence microscopy showed segmental granular IgG staining along the capillary loops in SMGN, whereas GMGN showed global staining. On light microscopy, SMGN showed segmental thickening of the glomerular basement membrane, with spike formation, whereas GMGN showed global lesions. The frequency of C1q deposits in SMGN was significantly higher than that in GMGN (91 versus 41%; P < 0.01). On electron microscopy, mesangial electron-dense deposits were detected in 10 (91%) cases of SMGN and also were found in the subepithelial and intramembranous area, whereas only six (22%) cases of GMGN had mesangial electron-dense deposits (P < 0.001). There were no significant differences in clinical features between the groups. Two children with SMGN underwent a repeat biopsy 3 yr after the first biopsy, and both patients again showed SMGN. At the final observation (mean observation time 7.5 yr in SMGN and 12.4 yr in GMGN), all children of both groups had a good outcome. In conclusion, these findings as a whole suggest that SMGN may be another glomerular disease entity with child predominance that is distinctive from GMGN. Clin J Am Soc Nephrol 1: 723–729, 2006. doi: 10.2215/CJN.01211005

embranous glomerulonephritis (MGN) is a chronic glomerular disease with nephrotic or nonnephrotic proteinuria (1,2). The characteristic histopathologic features of MGN are a diffusely thickened glomerular capillary wall that exhibits projections or spikes under light microscopy, fine granular staining for IgG and complement along the glomerular capillary wall on immunofluorescence microscopy, and electron-dense deposits (EDD) in the subepithelial area on electron microscopy (3). Although generally MGN is a global glomerular disease, patients who have idiopathic segmental MGN (SMGN) and IgG deposits in a portion of the glomerulus are relatively common. At present, it remains to be determined whether SMGN represents a discrete entity. To define the clinicopathologic significance of SMGN and to clarify whether SMGN is the same entity as global MGN (GMGN), we compared the two diseases with respect to clinical features and renal pathologic findings.

M

Materials and Methods

Received October 4, 2005. Accepted March 14, 2006.

Histopathologic Evaluation

Published online ahead of print. Publication date available at www.cjasn.org.

Renal tissue was obtained by needle biopsy under x-ray or ultrasound control. Renal biopsy specimens were examined by routine light, immunofluorescence, and electron microscopy. Biopsy specimens for light microscopy were fixed in phosphate-buffered 10% formalin; embedded in paraffin; sectioned at 4-␮m thickness; and stained with

Address correspondence to: Dr. Koichi Nakanishi, Department of Pediatrics, Wakayama Medical University, 811-1 Kimiidera, Wakayama City, Wakayama 641-8509, Japan. Phone: ⫹81-73-441-0633; Fax: ⫹81-73-444-9055; E-mail: [email protected] Copyright © 2006 by the American Society of Nephrology

Patients Children who underwent routine renal biopsies in our hospitals between September 1978 and August 2004 were analyzed retrospectively. Patients with systemic lupus erythematosus (SLE) and viral hepatitis specifically were excluded from our study. We divided patients with idiopathic MGN into two groups, SMGN and GMGN, according to the findings of renal immunofluorescence microscopy. In this study, “global” indicates a lesion involving the whole glomerulus, whereas “segmental” indicates a lesion involving a portion of the glomerulus (i.e., some capillary lumina remain uninvolved) (4). SMGN and GMGN were defined on the basis of immunofluorescence features: SMGN was characterized by segmental granular IgG staining along the capillary loops, whereas GMGN showed this feature in the whole glomerulus. Hematuria was defined as ⱖ5 erythrocytes/high-power field. Nephrotic syndrome was defined as urinary protein excretion ⱖ40 mg/h per m2 with hypoalbuminemia ⱕ25 g/L. Hypertension was defined as present when the systolic or diastolic pressure exceeded the upper normal limit for Japanese healthy children (mean ⫾ 2 SD).

ISSN: 1555-9041/104-0723

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hematoxylin-eosin, periodic acid-Schiff, periodic acid methenamine silver, and Masson trichrome. Tissue for immunofluorescence study was snap-frozen in acetone dry ice; cut at 3 ␮m; and stained with FITClabeled commercial antibodies to human IgG, IgA, IgM, C1q, C4, C3, and fibrinogen (Cappel, Aurora, OH). Immunofluorescence staining was graded as follows: Negative, 0; slight, 1⫹; moderate, 2⫹; and intense, 3⫹. The portion of tissue for electron microscopy was fixed in phosphate-buffered 5% glutaraldehyde for 2 h and postfixed for 1 h in 2% osmium tetroxide. It subsequently was processed through graded alcohol and embedded in Epon 812 resin. Ultrathin sections were cut on an LKB ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a JEM-100S electron microscope (JEOL, Tokyo, Japan). One or two glomeruli from each specimen were examined. Morphologic evaluation of MGN by electron microscopy was performed according to Ehrenreich and Churg four-stage classification as described previously (5).

Serial Section Study and Confocal Microscopy Observation We examined 10 serial sections (4 ␮m) from paraffin blocks in two patients from each group to confirm segmental and global status. The sections were stained by a direct method using horseradish peroxidase– conjugated goat anti-human IgG antibody (Cappel, MP Biomedicals, Aurora, OH). The sections were treated by proteinase K (0.4 mg/ml, 6 min at room temperature; Sigma, St. Louis, MO) for antigen retrieval. All glomeruli from kidney sections were photographed (⫻400) using an Olympus C4040 CCD camera in combination with an Olympus BX50 microscope (Olympus, Tokyo, Japan). The images were analyzed using ImageJ 1.34s (National Institutes of Health, Bethesda, MD) on a MacIntosh computer (Apple Japan, Tokyo, Japan). The outer margin of the glomerular tufts on each photograph was identified and traced manually, and the area thus encircled was measured. The positive anti–IgGstained glomerular basement membrane (GBM) and total GBM areas were measured manually, and the IgG-positive area ratio on each section was calculated by dividing the former by the latter (6). The global IgG-positive ratio on each section was calculated by dividing the number of glomeruli with global IgG-positive staining by the total number of glomeruli. Moreover, kidney sections (7 ␮m) from two other patients in each group were observed by a Radiance2100 confocal microscopy (BIO-RAD, Tokyo, Japan). The sections were stained with a FITC-labeled antibody to human IgG as described above. Seven serial images in a glomerulus were recorded every 1 ␮m thickness with autofocus system.

Clin J Am Soc Nephrol 1: 723–729, 2006

Statistical Analyses Results were analyzed with JMP 5.0.1J software (SAS Institute Inc., Cary, NC). The associations between categorical variables were examined by Fisher exact test. Continuous characteristics of the groups were compared with the Mann-Whitney U test. A two-tailed P ⬍ 0.05 was considered to be statistically significant.

Results Histopathologic Findings Between September 1978 and August 2004, 1604 children underwent first renal biopsy examinations in our hospitals. Among them, 38 children (2.4%; 25 boys and 13 girls) received a diagnosis of idiopathic MGN. The mean age at presentation in patients with idiopathic MGN was 7.6 yr (range 1.5 to 16.0 yr). Five patients with idiopathic MGN underwent repeat renal biopsies. Of the 38 children who received a diagnosis of idiopathic MGN, 11 (29%; eight boys and three girls) showed SMGN and 27 (71%; 17 boys and 10 girls) showed GMGN (Table 1). Sufficient renal biopsy tissue for histologic evaluation (minimum of 10 glomeruli) was available in all patients. The mean ⫾ SD number of glomeruli observed for light microscopy was 34.9 ⫾ 16.8 (range 12 to 70) in the SMGN group and 33.3 ⫾ 12.7 (range 10 to 65) in the GMGN group. The mean ⫾ SD number of glomeruli observed for immunofluorescence microscopy was 4.4 ⫾ 1.3 (range 2 to 5) in the SMGN group and 5.9 ⫾ 4.4 (range 2 to 15) in the GMGN group. Findings of immunofluorescence microscopy are shown in Table 2, and representative immunofluorescence micrographs are shown in Figure 1. Patients with SMGN had segmental granular IgG staining along the capillary loops, whereas patients with GMGN showed global granular IgG staining. Glomeruli in patients with SMGN showed a lesion involving 10 to 70% of the glomerulus, whereas the entire glomerulus was affected in patients with GMGN. The frequency of positive C1q capillary pattern deposits in the SMGN group was significantly higher than that in the GMGN group (10 [91%] of 11 patients versus 11 [41%] of 27 patients; P ⬍ 0.01). C1q deposits were observed in 1⫹ to 3⫹ amounts in 10 patients with SMGN. There was no significant difference in mean intensity of posi-

Table 1. Clinical features in patients with SMGN or GMGNa

Gender (M/F) Mean age at onset (yr; range) Mean period from onset to first biopsy (yr; range) No. (%) of patients showing hematuria No. (%) of patients showing nephrotic syndrome Mean creatinine clearance at first biopsy (ml/min per 1.73 m2) Mean urine protein excretion at first biopsy (g/d per m2) No. (%) of patients showing hypertension a

SMGN (n ⫽ 11)

GMGN (n ⫽ 27)

P

8/3 6.9 (1.5 to 14.8) 0.9 (0.1 to 2.0) 8 (73) 3 (27) 145

17/10 7.9 (3.0 to 16.0) 0.5 (0.0 to 2.7) 20 (74) 6 (22) 134

NS NS NS NS NS NS

1.1

1.0

NS

1 (9.1)

1 (3.7)

NS

GMGN, global membranous glomerulonephritis; SMGN, segmental MGN.


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Table 2. Immunofluorescence results in patients with SMGN or GMGN

Antigen

IgG IgA IgM C1q C4 C3 Fibrinogen a

Mean Intensitya of Positive Stain (Range) SMGN Patients (n ⫽ 11)

GMGN Patients (n ⫽ 27)

11 (100) 1.7 (1 to 3) 5 (45) 1.0 (1 to 1) 7 (64) 1.0 (1 to 1) 10 (91) 1.5 (1 to 3) 5 (45) 1.2 (1 to 2) 8 (73) 1.4 (1 to 3) 2 (18) 1.0 (1 to 1)

27 (100) 2.2 (1 to 3) 6 (22) 1.0 (1 to 1) 10 (37) 1.0 (1 to 1) 11 (41) 1.2 (1 to 2) 7 (22) 1.0 (1 to 1) 18 (67) 1.0 (1 to 1) 10 (37) 1.0 (1 to 1)

P

NS NS NS NS NS NS ⬍ 0.01 NS NS NS NS NS NS NS

Slight, 1⫹; moderate, 2⫹; intense, 3⫹.

tive C1q deposits between the groups. For the other antigens, there were no significant differences in positive frequency or mean intensity of positive deposits between the groups. In agreement with immunofluorescence findings, light microscopy showed that all patients with SMGN had segmental thickening of the GBM, with spike formation, whereas all patients with GMGN showed global lesions. Electron microscopy showed that patients with SMGN had segmental distribution of EDD located in the subepithelial area, whereas patients with GMGN had global distribution of EDD located in the subepithelial area. There was no difference in the size and the quality of EDD. The dominant morphologic stages that were revealed by electron microscopy in patients with SMGN or GMGN are shown in Table 3. There was no significant difference between the groups in the ratio of the dominant morphologic stage shown by electron microscopy. In addition, small and localized EDD were detected in the mesangial area in 10 (91%) patients with SMGN as well as in the subepithelial and intramembranous area (Figure 2). However, only six (22%) patients with GMGN had mesangial EDD. There was a significant difference in the frequency of positive mesangial EDD between the groups (91 versus 22%; P ⬍ 0.001).

Figure 1. Representative images of IgG staining by immunofluorescence in patients with segmental membranous glomerulonephritis (SMGN). The immunofluorescence images show segmental granular staining of IgG along the capillary loops (arrowheads indicate the contours of the glomeruli. Magnification, ⫻400.

Clinical Findings Table 3. Dominant morphologic stages by electron microscopy in patients with SMGN or GMGNa Stage

I II III IV

SMGN (n ⫽ 11)

GMGN (n ⫽ 27)

P

3 (27%) 5 (46%) 3 (27%) 0 (0%)

7 (26%) 14 (52%) 6 (22%) 0 (0%)

NS NS NS NS

The clinical features of both groups are listed in Table 1. The mean age of onset was 6.9 yr (range 1.5 to 14.8) in the SMGN group and 7.9 yr (range 3.0 to 16.0) in the GMGN group. All children had proteinuria. Three (27%) children in the SMGN group and six (22%) children in the GMGN group presented with nephrotic syndrome. There were no significant differences between the groups in gender ratio, mean age of onset, mean period from onset to first biopsy, proportion of patients who showed hematuria, proportion of patients who showed ne-

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in the SMGN group and 12.4 yr (range 0.5 to 23.7 yr) in the GMGN group. At the most recent examination, all patients in both groups showed normal urinalysis (six patients with SMGN and 18 patients with GMGN) or mild proteinuria (urine protein excretion ⬍0.5 g/d per m2; five patients with SMGN and nine patients with GMGN). No patients had clinical or laboratory evidence of SLE or mixed connective tissue disease during the follow-up period. Tests for anti-nuclear antibodies were always negative in all patients of both groups. Serum hepatitis B surface antigen was absent from all patients. None of the patients had been exposed to drugs or heavy metals that are associated with the occurrence of MGN.

Serial Section Study and Confocal Microscopy Observation

Figure 2. Representative electron microscopic image from a patient with SMGN. Electron-dense deposits (EDD) were detected in the mesangial area (arrows) as well as in the subepithelial and intramembranous area (arrowheads). Magnification, ⫻3000.

Table 4. Treatment in patients with SMGN or GMGNa

Discussion

Treatment

SMGN (n ⫽ 11)

GMGN (n ⫽ 27)

P

Yes/no Prednisolone Dipyridamole ACEI Chinese medicine (Sairei-to) Mizoribine

5/6 3 (27%) 1 (9%) 4 (36%) 0 (0%) 0 (0%)

18/9 9 (33%) 5 (19%) 4 (15%) 3 (11%) 1 (4%)

NS NS NS NS NS NS

a

Serial section study data are shown in Table 5. Representative pictures of serial sections in a patient with SMGN are shown in Figure 3. Representative pictures of confocal microscopy findings in patients with SMGN or with GMGN are shown in Figure 4. These findings demonstrate that segmental changes are not a product of incomplete glomerular examinations.

ACEI, angiotensin-converting enzyme inhibitor.

phrotic syndrome, mean creatinine clearance at first biopsy, mean urine protein excretion at first biopsy, or proportion of patients who showed hypertension. Treatment in patients with SMGN or GMGN are shown in Table 4. Five patients with SMGN and 18 patients with GMGN received medication. There was no significant difference in the treatment agents or frequency of treatment between the groups. Two patients with SMGN underwent a repeat renal biopsy 3 yr after their first biopsies, and both again showed SMGN. The mean follow-up period was 7.5 yr (range 1.1 to 16.6 yr)

It generally is thought that membranous transformation is global throughout all glomerular capillaries in idiopathic MGN (5). Therefore, although there are numerous papers on idiopathic MGN, at present we can find only a few that deal mainly with idiopathic SMGN (7,8), and most of these are case reports. In this study, we first describe a considerable number of children with idiopathic SMGN in a large series of renal biopsies and clarify the frequency of idiopathic SMGN. We had 38 children with idiopathic MGN from a total of 1604 children with first renal biopsies, and 11 of these patients (29% of the total with idiopathic MGN) showed SMGN. This frequency of 29% is strikingly high. Discussion of frequency requires care with definitions of conditions. In this study, we defined segmental as a lesion involving a portion of the glomerulus (i.e., some capillary lumina remain uninvolved), whereas global was defined as a lesion involving the whole glomerulus by immunofluorescence microscopy (4). Whether the high frequency of SMGN in our study is caused by our definition of the condition or by ethnic or geographic factors is unknown. It is possible that idiopathic SMGN may be more common than generally thought. More extensive and careful studies may elucidate the situation.

Table 5. Serial section study data in patients with SMGN or GMGN Patient

SMGN 3036S 2395S GMGN 0980D 6374D

Range of Glomeruli Number

Mean Global IgG-Positive Ratio (Range)

Mean IgG-Positive Area Ratio (Range)

28 to 34 43 to 49

27.3% (14.7 to 42.4%) 23.6% (12.2 to 37.5%)

63.8% (53.6 to 75.9%) 51.2% (42.5 to 64.1%)

42 to 48 31 to 34

100% 100%

100% 100%



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Figure 3. Representative serial section images of IgG staining by light microscopy in a patient with SMGN. (A) The start section. (D) The last section. Magnification, ⫻400 (hematoxylin counterstain).

We defined SMGN on the basis of immunofluorescence findings. Because mild changes in idiopathic MGN may not be evident only by light microscopy, which may result in misdiagnosis of mild GMGN as SMGN, we confirmed the segmental status by immunofluorescence microscopy. Our study is the first to show a difference not only in quantity but also in quality between the lesions of the segmental and global forms of idiopathic MGN. Although there were no significant differences in clinical features between the two groups, there were significant differences in the frequency of positive C1q staining and mesangial EDD. We have experienced 11 children with idiopathic SMGN, and 10 (91%) of them had predominant C1q deposits, in contrast to the GMGN group. In addition, the deposits were seen by electron microscopy in the mesangial areas as well as in the subepithelial areas in 10 (91%) patients of the SMGN group. Although intense staining for C1q should arouse suspicion of SLE (3,9), no patient in our study had clinical symptoms or laboratory investigations suggesting SLE. Although SLE may develop eventually in certain patients with SMGN (9), there was no such case in our group of patients with SMGN after a mean follow-up period of 7.5 yr (range 1.1 to 16.6 yr). Another point in which our SMGN group differs from the general lupus MGN population is gender ratio. Jennette et al. (9) reported that the male/female ratio of a nonlupus MGN group was 2:1 and that of a lupus MGN group was 1:4. Our SMGN group showed a clear male predominance (approximately 3:1), similar to that of our GMGN group (approximately 2:1). However, the follow-up periods in some patients in

our study are relatively short. Therefore, we may have missed the opportunity to capture positive serology for SLE. It is interesting that there is one finding in common between patients with SMGN in previous reports and our patients with SMGN, which is that idiopathic SMGN shows a child predominance. Gaffney et al. (7) and Yamamoto et al. (8) reported four and three child cases with idiopathic SMGN, respectively. Habib et al. (1) also described a similar lesion in children in their series. A different host immune response in children may cause SMGN. Most cases of adult-type idiopathic MGN occur in individuals who are older than 40 yr and show a global pattern. Meanwhile, most cases of the child type ameliorate before the patients reach adulthood. Therefore, there also is a possibility that adult- and child-type idiopathic MGN belong to different categories. In the same study period, we diagnosed MGN in 41 adults, but only one patient showed SMGN. One might speculate that SMGN is an incomplete or atypical form of idiopathic MGN. However, the differences in qualitative aspects, namely C1q deposits and mesangial EDD, between the groups suggest that SMGN is a distinct entity. Similarly, SMGN might be considered as an early stage of idiopathic MGN. Yamamoto et al. (8) reported that although the mean age of onset of SMGN in three children was less than that in patients with GMGN, two of their patients with SMGN also showed segmental staining of IgG in serial renal biopsies as well as in the first biopsy. Our data show no significant difference in mean age of onset between the two groups; moreover, two patients in our SMGN group received serial renal biopsies

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Figure 4. Representative images of IgG staining by confocal immunofluorescence microscopy in two patients with SMGN and in a patient with global MGN (GMGN). The first, third, fifth, and seventh pictures of a glomerulus in each patient are shown. (A through D) A glomerulus with ⬍50% IgG-positive area in a patient with SMGN. (E through H) A glomerulus with ⬎50% IgG-positive area in a patient with SMGN. (I through L) A glomerulus with 100% IgG-positive area in a patient with GMGN. Magnification, ⫻400. that again showed SMGN. Therefore, it seems unlikely that SMGN is an early stage of idiopathic MGN. It might be thought that SMGN is a milder form of idiopathic MGN. However, our data show no significant difference between the groups in mean urine protein excretion at first biopsy or in the percentage of patients who show nephrotic syndrome. The prognosis generally is known to be favorable in child idiopathic MGN (2,3). At most recent follow-up, all patients in both of our groups showed normal urinalysis (six patients with SMGN and 18 patients with GMGN) or mild proteinuria (five patients with SMGN and nine patients with GMGN) and had not developed end-stage disease. We cannot show a difference in prognosis between the groups, although in previous reports (1,7), children with SMGN had a favorable clinical course. Moreover, our pathologic data reveal no significant difference between the groups in the ratio of the dominant morphologic stage on electron microscopy (Table 3). The segmental state may not modify the clinical course of idiopathic MGN. Conversely, the data scale in our study may not be enough to detect the prognostic difference between the groups. Furthermore, a larger series may prove the segmental lesion to be more benign. In this study, five (45%) patients with SMGN and 18 (67%) patients with GMGN received medication (Table 4). There was no significant difference in the treatment agents or frequency of treatment between the groups. There are no controlled clinical trials in children of treatment, and no definitive approach to

therapy is available at this time (3). There also is no sufficient information to determine the frequency of spontaneous remission in a child who has idiopathic MGN and is not treated. Therefore, it is difficult to evaluate whether different (milder or more severe) treatment is adequate for SMGN. One might speculate that SMGN is idiopathic MGN with partial resolution. Gaffney et al. (7) reported that in two of four patients with SMGN, previous biopsy specimens had shown typical stage II and stage III MGN, indicating that at least in some cases, SMGN represents partial resolution of MGN. Bertani et al. (10) also described a 3-yr-old boy who had hereditary nephritis and showed focal SMGN in the first biopsy and in whom the complete absence of EDD and minimal immunofluorescence findings were observed in the second biopsy, 3 yr after the first. It has been suggested that idiopathic MGN in adults is characterized by deposition of the predominantly IgG4 subclass (11–14). The characteristics of IgG4 may explain a generally observed finding in adult idiopathic MGN of abundant C3 with little C1q deposition, which reflects activation of the alternative, rather than classical, complement pathway (11). Although to date there has been no report of IgG subclass in child idiopathic MGN, it may be interesting to compare IgG subclass between SMGN and GMGN in children. Further investigation of this condition seems warranted.



Conclusion Our findings as a whole suggest that SMGN may be another glomerular disease entity with child predominance, although further extensive studies are needed to confirm the existence of a segmental form of idiopathic MGN. The accumulation of careful studies that are based on the concept that SMGN may be another glomerular disease may establish a new disease entity that is different from GMGN.

7.

8.

Acknowledgments A part of this study was presented at the 37th annual meeting of the American Society of Nephrology, October 29 to November 1, 2004, St. Louis, MO, and was published in abstract form (J Am Soc Nephrol 15: 343A, 2004).

9.

10.

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