Journal of Tongji Medical University ~ ~= ~ ~ �9 �9 �9 ( #b 3~ I~ ) 16 (3):173 -178, 1996 173

Immunohistological Study on the Mechanism of Mesangial Proliferation

ZHANG Ming ( ~ w)~)-, GUO Muyi ( ~ f g ) " ", ZHANG Xiurong ( ~ ) " ", JIN Huiming (~,gtd;)"

" Department o f Pathophysiology, Shanghai Medical University, Shanghai, 200032 �9 "Department o f ~athology, Shanghai Medical University, Shanghai 200032

Summary: Inflammatory cells infiltrated in glomeruli and proliferating glomerular cells were immunohistochemically studied in 87 renal biopsies from patients with glomerulonephritides (GN) of various types, by using monoclonal antibody to Mac387 and PCNA (proliferative cell nuclear antigen) respectively. The results showed that the expressions of Mac387 and PCNA were generally increased in proliferative GN, compared with non-proliferative GN. The expression of Mac387 was related to the severity of mesangial cell proliferation determined by fight mi- croscopy, but the increase of PCNA expression was found in severely proliferative GN. Although certain relationship between Mac387 and PCNA expression on glomerular cells was observed, it was not statistically significant. Finally, the possible mechanism of glomerular mesangial proliferation was discussed. Key words: mesangial proliferation; glomerulonephritis; immunohistochemistry

Mesangial proliferation is one of the common pathologic features of glomeru- lonephritides ( G N ) of various types , in- eluding IgA nephropathy , Schonlein- Henoch syndrome, membranoproliferat ive G N , mesangial proliferative G N , lupus nephri t is , and so on. The mechanism of mesangial proliferation has not been clari- fied completely. It has been showed that the mechanism of mesangial proliferation is very complicated. It often involves the cytokines or growth factors , such as interleukin-1 (IL-1)E~1, interleukin-6 ( IL-6 )Ezl and platelet derived g rowth factor (PDGF) E3"93, which were derived from intrinsic glomeru- lar cells, including glomerular mesangial cells and endothelial cells, or from infiltrat- ing inf lammatory cells and platelets , acting in an endocrine, autocrine or paracrine fash- ion C~'sl. In order to understand the cellular biological mechanism of mesangial prolifera- t ion, glomerular immunoglobulin ( I g ) de~ posit ion, infiltration of inf lammatory cells, proliferation of mesangial cells and the ~ela- tionships among them were analyzed using

ZHANG Ming, male, born in 1966, Lecturer

immunohistochemical method in 87 patients with GN of various types.

1 MATERIALS A N D M E T H O D S

1. 1 Patients For this s tudy , renal biopsy specimens

from 87 patients with primary or secondary GN were collected from the nephrology de- par tments of Zhongshan Hospi ta l , Huashan Hospital and Children Hospital affiliated with Shanghai Medical Universi ty during April 1990 to April 1993. The patients con- sisted of 37 males and 50 .females. The age ranged from 7 to 58 years with average be- ing 25. 2. In all cases, a definite diagnosis was made on the basis of the WHO diagnos- tic standards of GN (1982). They included 11 cases of mesangial proliferative G N , 10 cases of membranoproliferative G N , 3 cases of crescentic G N , 8 cases of Schonlein- Henoch syndrome, 13 cases of lupus nephri- t is , 24 cases of IgA nephropa thy , while the other 18 cases of non-proliferative G N , in- cluding 12 cases of gtomerular minor lesion, 6 cases of membranous G N , seved as con- trols.

174 Journal of Tongji Medical University 16 (3): 173-178, 1996

1.2 Renal Biopsy 1.2. 1 Routine Pathological Examination

The specimens were routinely pro- cessed for light, immunofluorescent and electron microscopy. For light microscopy, the sections were stained with hematoxylin- eosin, periodic acid-schiff, periodic acid- schiff methenamine silver. The frozen sec- tions for immunofluorescent microscopy were stained by FITC-labelled antihuman IgG, IgA, IgM, C3 with 0 -- 3 degree to express the intensity of fluorescence. Tis- sues for electron microscopy were fixed im- mediately upon procurement in 3 % glu- taraldehyde and followed by post-fixation in 1 ~ osmic acid and dehydrated with graded alcohol, and then the specimen was embed- ded in Epon. Ultra-thin sections were dou- ble-stained with uranyl acetate and lead cit- rate and examined with H-550 transmission election microscope. 1 .2 .2 Immunohistochemicai Studies Reagents used included primary antibodies: mouse anti-human proliferative cellular nu- clear antigens ( P C N A , Oncogene Science Inc. , U S A ) , mouse anti-human Mac387 (Programa Inc. , U S A ) monocl0nal anti- bodies, with the working dilution being 1: 20 and 1:100 separately and ABC reagents kit (Vector Inc. , USA).

Immunoperoxidase staining procedure involved use of a sensitive avidin-biotin complex (ABC) technique. Briefly, 3 ttm paraffin embedded sections were deparaf- fined, fixed and incubated with above-men- tioned primary monoclonal antibodies for 1 h, biotinated horse anti-mouse im- munoglobulin (1: 100) for 1 h and ABC complex (1: 100) for 1 h sequentially, fol- lowed by 3'-5'-diamino-benzidine (DAB) and hydrogen peroxide for 3 to 5 min. Sec- tions stained for Mac 387 were counter- stained with hematoxylin. 1.2. 3 Quantitation The degrees of mesangial proliferation with HE stained was graded under the light microscope as fol- lows : Grade I: the morphology of glomeru[i was approximately normal with only slight mesangial extracellular matrix expansion or focal and segmental mesangial cell prolifera- tion detected. Grade II: moderate focal and segmental, mesangial cell proliferation or

slight diffuse mesangial cell proliferation. Grade III: moderate diffuse mesangial cell proliferation accompanied by few glomeru- lar crescentic formation. Grade IV: severe mesangial cell proliferation accompanied by many glomerular crescentic formation.

For each specimen, the number of glomeruli per section was counted, in which the PCNA or Mac 387 positive cells were counted correspondingly. The mean values of the positive cell per glomerulus were cal- culated. 1.3 Statistical Analysis

The number of positive cells per glomerular cross-section obtained with the different McAb in the various groups were compared by using a Wilcoxon Rank Sum test, because the data were not normally distributed and were too small to allow crit- ical assessment of possibility of the logarith- mic transformation resulting in a truly nor- mal distribution. Correlation between two indices was determined using Spearman's test. P < 0 . 05 was considered to be of sta- tistically significance.

2 RESULTS

2. 1 The Expression of Mac387 With immunoperioxide staining,

Mac387 positive cells appeared dark brown in the cytoplasm without the blank nuclear area, rendering oval, spindle-shaped or ir- regular forms. The positive cells were lo- cated in the capillaries of glomerular mesan- gial areas, in or on the Boman's capsules and crescents. They were also distributed sparsely or focally in the interstitia of kid- ney.

The mean values of the Mac 387 posi- tive cell per glomerulus in various types of GN were shown in fig. 1. It was increased in the following types of GN in the order of glomerular minor lesion, membranous GN, IgA nephropathy, Schonlein-Henoch syn- drome, mesangial proliferativ e GN, lupus nephritis and crescentic nephritis.

By comparing the proliferative GN ~vith non-pr01iferative GN, it was demonstrated that, except Schonlein-He.noch syndr0me, the Mac387 positive values in various types of proliferative GN were significantly , higher

ZHANG Ming et al . Mechanism of Mesangial Proliferation 175

than those in control group ( P < 0 . 0 5 , table 1).

Table 1 Comparison of Mac387 infiltration between non-proliferative and proliferative GN

Types n T value P value

Nonproliferative 18 Glomerular minor lesion 12 Membranous GN 6

Primary GN Membranoproliferative GN 10 Mesangial proliferative GN 11 Crescentic GN 3

Secondary GN Scholein-Henoch syndrome 8 Lupus nephritis 13

IgA nephropathy 24

55 0 95 0.0017 6 0. 0018

59 0.0749 91 0

254.5 0.0115

2. 2 The Express ion of PCNA In each type of G N , the mean values of

PCNA positive cell per glomerulus were re- markably lower than those of Mac 387. Most PCNA positive cells were located in the mesangial area or on the Boman's cap- sule (including c rescen t s ) , imparting oval or global forms. Some of tubular epithelial nuclei were also positively stained.

The mean values of the PCNA-posit ive cell per glomerulus in various types of GN were shown in fig. 2. It was increased in

the following types of GN in the order of membranous G N , glomerular minor lesion, IgA nephropa thy , mesangial proliferative G N , lupus nephri t is , membranopro- liferative G N , Schonlein-Henoch syndrome and crescentic nephritis.

By comparing the proliferative GN with non-proliferative G N , it was demonstrated that except IgA nephropathy the P CN A positive values in various types of prolifera- tive GN were significantly higher than those in control group ( P dO. 05, table 2).

Table 2 Compar!son of PCNA infiltration between nonproliferative and proliferative GN

Types n T value P value

Nonproliferative 18

Glomerular minor lesion 12

Membranous GN 6

Primary GN

Memhranopi'oliferative GN 10

Mesangial proliferative GN 1 i Cescentic GN 3

Secondary GN

Seholein-Henoch syndrome 8

Lupus nephritis 13

IgA nephropathy 24

100 0.0311

101 0.0031

6 0.0015

58 0.0042

121 0.005

321 0.1086

2. 3 Relat ionship between Expression of Mac387 , PCNA and Glomerular lg Deposi- tion

The infiltration of Mac387-positive cells aggrandized with the fluorescent inten-

sity of IgG deposition increasing in each types of proliferative GN (Fig. 3 ) , while the expression of PCNA failed to show such relation. In 24 cases of IgA nephropa thy , the expressions of Mac 387 and PCNA had the tendency to decrease with the IgA depo-

176 Journal of Tongji Medical University 16 (3): 173-178, 1996

Q 10 o z

3.04

1.38 ~

6.9

MGN S H G N MPGN IgAGN. MsPN

).87

\ \ \ %\x \ \ \

CrGN LN

Fig. 1 Mac387 in different types of GN

~. 31 2. 67

"~ .2 \ x

Z ..90 \x \ \ ~-..~ 1 0.57 0.58 0.6 \ \ ! , NNN --'"

.~ 0 ML MsPN MPGN CrGN IgAGN LN SHGN

Fill 2 Pt NA in different typt.s ,,i (;N

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7

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Fig. 3

3.43

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2 3 Grade of IgG

Relationship between IgG & Mac387

8.14 .N

bl

sition in glomeruli increasing (fig. 4). 2 .4 Relationship between the Expression of Mac 387, PCNA and Glomerular Mesan- giai Cell Proliferation

The mean values of glomerular Mac 387-positive cells showed a significant cor- relation with the grades of mesangial cell proliferation ( P ~ 0 . 05, fig. 5). Though the mean value of PCNA positive cell in-

creased with the mesangial cell prolifera- tion, the statistical significance was showed only between grade III and IV of mesangial cell proliferation. 2 .5 Relationship between the expression of PCNA and Mac387

Generally speaking, among various types of GN the more Mca 387 positive cells infiltrated, the more PCNA expressed. However , in each type of GN, correlation test between the Mac 387 expression and PCNA expression of each case didn ' t show any statistical si~:nificance.

; • 3.5-

~ 2 . s-

~ 1 . 5 -

Fig. 4

3. 44

[ ] VCNA \ \ \ \ \ \ ~ Mac387

�9 " " 1.13

~ \ ~ 0.58

2 3 Grade of IgA

Relationship between "IgA & Mac387, PCNA

6 12

~ o

g,

Fig. 5

[ ' ] PCNA 10. 34 k \ \

[ ] Mac387 k \ \ k \ \

�9 ~ % \ \ % \ \

140"a..48.~ 0.. 4 3 ~ . O. 49 \ \ \

i 2 3 4 Grade of MsC

Relationship between MsC & Mac387, PCNA

3 DISCUSSION

Mac387 antibody is rased agains t high- ly purified human peripheral monocytes. In 1987, 'Flavell et a l E6J prepared the commer- cial a n t i b o d y which combined specifically

ZHANG Ming et al. Mechanism of Mesangial Proliferation 177

with human macrophage-monocytes. In the present study, Mac387 positive cells in vari- ous types of proliferative GN were detected using ABC immuno-enzyme method and were compared with those in non-prolifera- tive GN. It was demonstrated that except those of Schonlein-Henoch syndrome, Mac387 positive cells increased significantly and correlated with glomerular IgG deposi- tion and mesangial cell proliferation. The analysis of Mac387 positive celldistribution in various types of proliferative GN showed that the GN with acute and active clinical course, such as crescentic GN, lupus nephritis, membranoproliferative GN, had more infiltration of Mac387 positive cells, in which mesangial cell hypercellularity was also more serious. This suggested that the infiltration of blood-derived macrophages and granulocytes might be one of the impor- tant causes of mesangial hypercellularity. It was also found that there were a lot of Mac387 positive cells in crescents, which confirmed that monocytes played an impor- tant role in the formation of crescents. The reason that the Mac387 positive cell number of Scholein-Henoch syndrome didn't show significant difference as compared with that of control group might be that the number of cases was too few (8 cases) or they were in a static state. In IgA nephropathy, IgA deposition had the reverse tendency with the infiltration of Mac387 positive cells. One of the possible explanations to this phe- nomenon is that the macrophage-monocytes act as "scavenger" cells to phagocytosis of the immune complex in glomeruli c7].

Proliferative cellular nuclear antigen (PCNA) was identified as an auxiliary pro- tein of DNA polymerase-delta, which most likely involved in DNA repair. It increases in the cell nuclei during S phase, so it can be used as an indication of the DNA content in S phase and the cellular proliferation. The commercial monoclonal antibody (McAb) for PCNA can be applied to the paraffin-embedded tissue section and is proved better than 3H-thymidine incorpo- ration, bromodexyuridine (Brdu) incorpo- ration followed by staining with Anti-Brdu McAb and ki-67 detection with immunohis- tochemical technique on frozen section in re-

flection of the intensity of cell division and proliferation Is] , so it has been applied wide- ly to the study on proliferation and differen- tiation of tumor cells. Recently Floege et

a l [9] used it to determine the glomerular cell proliferation in mesangial proliferative GN. The present study detected the PCNA ex- pression in 69 cases of proliferative and 18 cases of non-proliferative GN and found that except IgA nephropathy, the PCNA expression in each type of proliferative GN was significantly higher than that in non- proliferative GN. PCNA was closely related to mesangial cell proliferation (especially in Grade III, IV) , which indicated that PCNA may be used as a marker of the glomerular mesangial cell proliferation. Why PCNA positive cells in IgA nephropathy didn't in- crease significantly compared with those in control group is not very clear. It might have something to do with the facts that its pathologic process was in a static state or that most of the cases belonged to minimal change type of IgA nephropathy.

The studies of recent year., ~l,,w~,d that the monocytes infiltrating the glomeruli could activate the mesangial cells through releasing manifold cytokines, including IL- l , PDGF etc. . to promote the DNA syn- thesis or proliferation of mesangial cells [~~ The results of the present study confirmed the role of infiltrating intrinsic cells. How- ever, our analysis of the relationship be- tween Mac387 and PCNA revealed that al- though among various types of GN or a- mong different grades of glomerular hyper- cellullarity, the high Mac387 positive types or grades have more PCNA expressed, in each type of GN, there was no correlation between Mac387 and PCNA for each ease, i. e . , the ease with more macrophage-mono- cyte infiltration was not the case with more cellular proliferation at the same time. Some possible explanations are as follows: 1) In recent years, it was demonstrated in the animal model of proliferative GN that it was the macrophage-monocyte infiltration that was mainly responsible for the hyper- cellularity in the early period of disease. With the development of the disease, they could dissipate gradually, while the cy- tokines secreted by these cells activate the

178 Journa! of Tongji Medical University 16 (3) .. 173-178, 1996

intrinsic mesangial cells and the intrinsic cell proliferation plays a main part at the late time. With the cellular proliferation, large amount of ECM was produced. Hence, it was impossible that macrophage and PCNA increased in the same biopsy tis- sues of clinical renal acupuncture. It was more realistic that in the biopsy tissue of the early period proliferative GN, the Mac387 positive value increased, while in the middle or late period of proliferative GN, P C N A expressed highly. 2) Mesangial cell proliferation is regulated by multi tude elements, monocyte and granulocyte are on- ly one of the elements that cause mesangial cell proliferation. In addition, some cy- tokines paracrined or autocrined by lympho- cytes , platelets and glomerular intrinsic cells showed promoting effects, while oth- ers had inhibitory functions.

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(Received June 16, 1995)