Local macrophage proliferation in human glomerulonephritis

NIANSHENG YANG, NICOLE M. ISBEL, DAVID J. NIKOLIC-PATERSON, YOUJI LI, RENGAO YE,ROBERT C. ATKINS, and HUI Y. LAN

Department of Nephrology, Monash Medical Centre, Clayton, Victoria, Australia, and Department of Nephrology, First AffiliatedHospital of Sun Yat-Sen University of Medical Sciences, Guangzhou, People’s Republic of China

Local macrophage proliferation in human glomerulonephritis.Background. Local macrophage proliferation has been described in

several animal models of glomerulonephritis (GN), but its significance inhuman disease is unknown.

Methods. Double immunostaining for CD68 and the proliferating cellnuclear antigen (PCNA) was used to identify macrophage proliferation in84 biopsies from a variety of glomerulonephridities.

Results. A small resident population of glomerular and interstitialCD681 macrophages was identified in normal human kidney, of whichonly 1 to 2% showed evidence of proliferation on the basis of PCNAexpression. A mild macrophage infiltrate, with only occasional proliferat-ing macrophages, was seen in the less aggressive forms of GN (minimalchange disease, non-IgA mesangioproliferative GN and IgA nephropa-thy). This was in sharp contrast to the more aggressive forms of disease(lupus class IV, vasculitis-associated GN, crescentic GN and mesangio-capillary proliferative GN), in which the prominent macrophage infiltratescontained many proliferating macrophages, accounting for 28 to 47% ofthe total macrophage population. Macrophage proliferation was largelyrestricted to areas of severe tissue damage (glomerular segmental prolif-erative lesions, crescents and foci of tubulointerstitial damage), suggestingthat local proliferation is a mechanism for amplifying macrophage-mediated injury. Glomerular and interstitial macrophage proliferationgave a significant correlation with loss of renal function (P , 0.0001) andhistologic lesions (P , 0.0001), but not with proteinuria. Interstitial T-cellproliferation also gave a significant correlation with loss of renal functionand histologic damage, even though proliferation within the T-cell popu-lation was much lower than in the macrophage population.

Conclusions. This study demonstrates that macrophage proliferation isa feature of the more aggressive forms of human GN. Local proliferationmay be an important mechanism for amplifying macrophage-mediatedrenal injury. In addition, the degree of local macrophage proliferation maybe a useful diagnostic and prognostic indicator for human GN.

Glomerular and interstitial macrophage accumulation isa feature of almost all forms of human glomerulonephritis(GN), and the intensity of the macrophage infiltrate corre-lates with loss of renal function and histologic damage atthe time of biopsy and is a predictor of renal outcome[1–4]. Direct evidence that macrophages cause renal injuryhas come from animal studies in which a variety of anti-

macrophage strategies have been shown to inhibit theinduction or progression of renal injury in experimentalGN [5–10].

There has been much interest in the mechanisms bywhich macrophages accumulate within the kidney duringdisease. The entry of blood monocytes into the injuredkidney is regulated by a variety of chemokines and adhesionmolecules [11–13]. An additional mechanism contributingto macrophage accumulation and renal injury in experi-ment disease is local proliferation [14]. High levels of localmacrophage proliferation, identified by the presence ofmitotic figures, incorporation of bromodeoxyuridine andexpression of the proliferating cell nuclear antigen(PCNA), have been demonstrated in models of severerenal injury [15, 16]. The degree of local macrophageproliferation correlates with the severity of renal injury,being prominent in rat crescentic anti-glomerular basementmembrane (GBM) GN and acute allograft rejection, butonly occasionally seen in models featuring mild renal injury[15–20]. Local proliferation is largely restricted to areas ofsevere tissue damage, such as glomerular segmental prolif-erative lesions, crescents and granulomatous lesions [21].Thus, local proliferation has been postulated as a mecha-nism for amplifying macrophage-mediated tissue injury.

While there is good evidence to support the presence oflocal macrophage proliferation in experimental diseasemodels, the significance of these observations to humanGN remain to be established. The aim of this study was,therefore, to seek evidence of macrophage proliferation ina range of human glomerulonephridities.

METHODS

Patients

A total of 83 biopsies from the Department of Nephrol-ogy, Monash Medical Centre and the Department ofNephrology, First Affiliated Hospital of Sun Yat-Sen Uni-versity of Medical Sciences were examined (48 males and35 females; average age 42 years, range 13 to 82). Diseasecategories were as follows: minimal change disease (MCD,N 5 9), non-IgA mesangial proliferative GN (non-IgAMPGN, N 5 12), IgA nephropathy (IgAN, N 5 19),

Key words: macrophage, proliferation, T cell, PCNA, Ki-67, glomerulo-nephritis, granulomatosus lesions, renal injury, crescents.

Received for publication October 11, 1997and in revised form February 11, 1998Accepted for publication February 11, 1998

© 1998 by the International Society of Nephrology

Kidney International, Vol. 54 (1998), pp. 143–151

143

membranous GN (memb GN, N 5 4), focal glomerularsclerosis (FGS, N 5 10), lupus nephritis WHO Class II(lupus II, N 5 5), lupus WHO class IV (lupus IV, N 5 5),crescentic GN (cres GN, N 5 9; defined as .50% cres-cents, excluding IgAN and lupus), nephritis-associated withvasculitis (vasc GN, N 5 5; with ,50% crescents), mesan-giocapillary proliferative GN (MCPGN, N 5 5). All pa-tients were diagnosed on the basis of biopsy examination.Measurements of 24-hour protein excretion, serum creati-nine, and creatinine clearance were taken for each patientat the time of biopsy. Morphologically normal humankidney was obtained from the unaffected areas of five renaltumor nephrectomy specimens and used as control tissue inthis study.

Histopathology

Formalin-fixed, paraffin-embedded biopsy tissue sections(4 mm) were stained with hematoxylin and eosin. Thepercentage of glomeruli exhibiting segmental proliferativelesions or crescents was scored in 10 to 30 glomerularcross-sections (gcs) per biopsy. Glomerular hypercellularitywas assessed as follows: 0 5 normal (less than 60 cells/gcs);1 5 mild (60 to 90 cells/gcs); 2 5 moderate (90 to 120cells/gcs); 3 5 severe (more than 120 cells/gcs). The percentof the tubulointerstitial area exhibiting tubulitis, tubulardilation, atrophy, or fibrosis was assessed on H&E stainedsections. All scoring was performed on blinded sections.

Antibodies

Monoclonal antibodies (mAb) purchased from Dako(Glostrup, Denmark) were: KP1, mouse anti-CD68 presentin lysosomes of monocytes and macrophages [22]; UCHL1,mouse anti-CD45RO recognizing mature, activated T-cellsand a subset of resting T-cells [23]; PC10, mouse anti-proliferating cell nuclear antigen (PCNA), which recog-nizes cells in the G1, S, and G2 phases of the cell cycle [24,25]; MIB-1, mouse anti-Ki-67 antigen, which recognizescells in the G1, S, G2 and M phases of the cell cycle(Immunotech, Marseille, France) [26]. Peroxidase- andalkaline phosphatase-conjugated goat anti-mouse IgG,mouse peroxidase anti-peroxidase complexes (PAP), andmouse alkaline phosphatase anti-alkaline phosphatasecomplexes (APAAP) were purchased from Dako.

Immunohistochemistry

Two-color immunohistochemistry staining used a micro-wave-based technique [27]. Briefly, paraffin sections (4 mm)of formalin-fixed tissues were dewaxed, treated with 10minutes of microwave oven heating in 0.01 M sodium citratebuffer (pH 6.0) at 2450 MHz and 800 watts power. Thistreatment was used to retrieve the cytoplasmic antigenrecognized by the KP1 mAb, but was not used in detectionof the UCHL1 antigen. Slides were then washed twice inPBS, preincubated with 10% fetal calf serum (FCS) and10% normal goat serum in PBS for 20 minutes, drained andincubated with KP1 (1:1000) or UCHL1 (1:200) mAb at

4°C overnight. After washing in PBS, endogenous peroxi-dase was inactivated by incubation in 0.3% H2O2 in meth-anol, and sections were then incubated with peroxidase-conjugated goat anti-mouse IgG followed by mouse PAP

Fig. 1. Renal function data of the patient population. Measurements of (A)serum creatinine, (B) creatinine clearance, and (C) urinary protein excretion,are shown for the different disease types. Data are shown as mean 6 SEM.

Yang et al: Macrophage proliferation in human GN144

complexes and developed with 3,3-diaminobenzidine toproduce a brown color. At this point, slides were treated byanother round of microwave heating in order to denatureIgG molecules bound to the tissue and thus prevent an

antibody cross reaction and to improve antibody access tonuclei. Following a second preincubation step as above,sections were labeled with the PC10 mAb (1:1000) at 4°Covernight. After washing in PBS, sections were labeled

Fig. 2. Immunohistochemistry detection of proliferating cells in a case of crescentic glomerulonephritis. (A) Detection of proliferating cells (darknuclei) within the glomerular tuft and many proliferating cells within the crescent using the PC-10 (anti-PCNA) mAb. (B) A very similar result showingproliferating cells (dark nuclei) within the glomerular tuft and many proliferating cells within the crescent is seen using the MIB-1 (anti-Ki-67) mAb.Sections were counterstained with PAS. Magnification 3200.

Yang et al: Macrophage proliferation in human GN 145

sequentially with alkaline phosphatase-conjugated goat an-ti-mouse IgG and mouse APAAP complexes and devel-oped with Fast Blue BB Base (Sigma Chemical Co., St.Louis, MO, USA) to produce a blue color. Sections werecounterstained with periodic acid-Schiff reagent withouthematoxylin.

For detection of the Ki-67 antigen, sections were micro-wave oven heated, digested with 0.1% trypsin for 20minutes at 37°C in 0.1 M sodium acetate pH 4.0, washed inPBS, incubated with the MIB-1 (1:100) mAb overnightfollowed by with peroxidase-conjugated goat anti-mouseIgG followed by mouse PAP complexes and developed with3,3-diaminobenzidine.

Quantitation of immunohistochemical staining

Antibody stained cells in tissue sections were quantitatedas previously described [21]. Briefly, the number of singleand double stained cells were counted in 10 to 30 gcs perbiopsy. The number of single and double stained cells in thecortical interstitium were counted in at least 20 consecutivehigh power fields by means of a 0.02 mm2 graticule fitted inthe eyepiece of microscope, avoiding only glomeruli andlarge vessels. All scoring was performed on blinded slidesand the data are presented as the mean 6 SEM per gcs ormm2.

Statistical analysis

The number of macrophages, T cells and subsets ofproliferating cells in disease groups were compared to thenormal group using the Mann-Whitney U-test. In addition,the number of macrophages, T cells and subsets of prolif-erating cells were compared with measurements of renalfunction by the Pearson single correlation coefficient andwith measurements of histologic damage by the Spearman’srank coefficient.

RESULTS

Renal function in patient population

Measurements of serum creatinine, creatinine clearanceand urinary protein excretion are shown in Figure 1.Patients with less aggressive forms of GN (MCD, Non-IgAMPGN, IgAN and memb GN) had, on average, a normalrate of creatinine clearance (mean 105, range 64 to 184). Incontrast, those patients with more aggressive forms of GN

(lupus IV, vasc GN, cres GN and MCPGN) had a signifi-cant reduction in creatinine clearance (mean 33, range 2 to98). Proteinuria was variable between the diseases, beingheaviest in the memb GN group.

Macrophage proliferation in human glomerulonephritis

Proliferating cells in renal biopsies were detected byimmunostaining for PCNA and Ki-67 antigens, both ofwhich are expressed in the G1, S, G2 and M phases of thecell cycle [24–26]. Immunostaining of PCNA required 10minutes of microwave oven heating to optimize antigenretrieval. In contrast, immunostaining of the Ki-67 antigenwas more complex, requiring microwave treatment follow-ing by trypsin digestion to achieve optimal detection of theantigen. Both antibodies stained a similar number of cellsthat had a very similar distribution pattern, as illustrated inFigure 2. However, staining with the MIB-1 mAb (anti-Ki-67) was variable between staining runs, presumably due toa variation in the protease digestion step. Therefore, weused detection of PCNA as the marker of cell proliferationin this study.

Double immunostaining of normal human kidney andthe less aggressive forms of human GN (MCD, non-IgAMPGN, IgAN, memb GN) showed the presence of smallnumbers of macrophages within glomeruli, but only occa-sional proliferating macrophages (Fig. 3A and 4A). In-creasing numbers of glomerular macrophages were seen inFGS through to MCPGN, with marked glomerular macro-phage proliferation evident in aggressive forms of GN(lupus IV, vasc GN, cres GN and MCPGN). Indeed,proliferating macrophages accounted for between 29 and47% of total glomerular macrophages in aggressive formsof GN (Fig. 4A). Interstitial macrophage accumulation wasevident in most types of GN examined, being most markedin the more aggressive diseases (Fig. 4B). Few interstitialproliferating macrophages were seen in the less aggressiveforms of GN; however, interstitial macrophage prolifera-tion was prominent in aggressive forms of disease in whichthey accounted for 28 to 44% of the total macrophagepopulation (Fig. 4B).

Local macrophage proliferation was largely restricted to

Fig. 3. Double immunohistochemistry staining of macrophage proliferation in human glomerulonephritis. Paraffin sections were stained for KP11macrophages (brown) in combination with PCNA (blue nuclei) and counterstained with PAS. Examples of non-proliferating macrophages(KP11PCNA2 cells) are indicated by arrows, and examples of proliferating macrophages (KP11PCNA1 cells) are indicated by arrowheads. (A)Minimal change disease showing a single glomerular KP11 macrophage and little cell proliferation. (B) Mesangiocapillary proliferative GN showingprominent glomerular macrophage accumulation, with many of the macrophages proliferating, contributing to severe glomerular hypercellularity. (C)Lupus nephritis class IV showing substantial local macrophage proliferation within a glomerular segmental proliferative lesion. (D) Crescentic GNshowing macrophage proliferation within the glomerular tuft and in the crescent. (E) Crescentic GN showing high levels of macrophage proliferationwithin a focal area of tubulointerstitial damage. (F) Vasculitis-associated GN showing macrophage proliferation and giant cell formation within aperivascular granulomatous lesion. Note PCNA staining within the giant cell (asterisk). Magnification 3400. The publication of this figure in color wasmade possible by a grant from Janssen Cilag Pty Ltd.™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3

Yang et al: Macrophage proliferation in human GN146

Yang et al: Macrophage proliferation in human GN 147

areas of focal tissue damage. Many proliferating macro-phages were seen in areas of severe glomerular hypercel-lularity, glomerular segmental proliferative lesions, glomer-ular crescents, focal tubulointerstitial lesions and ingranulomatous lesions in which multinucleated giant cellsalso expressed PCNA (Fig. 3B-F). It should be emphasizedthat while high levels of macrophage proliferation wereevident within focal lesions, macrophages comprised only aminor population of the total number of proliferating cells(Fig. 3). Most proliferating cells were intrinsic renal cells,particularly tubular epithelial cells.

There was a substantial variation in the number ofproliferating macrophages in the different biopsies, eventhose within the same disease type. As this variationappeared to reflect the degree of histologic damage, weexamined the relationship between macrophage prolifera-tion and renal function and histologic injury. As summa-rized in Table 1, total glomerular macrophages and prolif-erating glomerular macrophages gave a highly significantcorrelation with serum creatinine, creatinine clearance andglomerular lesions. There was also an excellent correlationbetween total interstitial macrophages and proliferatinginterstitial macrophages with serum creatinine, creatinineclearance and tubulointerstitial lesions. However, glomer-ular and interstitial macrophage populations did not corre-late with proteinuria.

T-cell proliferation in human glomerulonephritis

Occasional T-cells were seen in the glomerulus and theinterstitium of normal human kidney, but no proliferatingT-cells were detected in either compartment (Fig. 5). Smallnumbers of glomerular T-cells were seen in most forms ofhuman GN. This was highly variable between biopsies suchthat increase in glomerular T-cell numbers failed to reachstatistical significance, except in crescentic GN (Fig. 5A).Occasional proliferating T-cells were observed in diseasedglomeruli, which accounted for between 4 and 17% of the

total T-cell population (Fig. 5A). In contrast, a markedinterstitial T-cell infiltrate was evident in all types of GNexamined. However, despite the high levels of T-cell accu-mulation within the interstitium, few proliferating T-cellswere seen. Proliferating T-cells accounted for only 1 to 9%of the total interstitial T-cell population (Fig. 5B). Similarto proliferating macrophages, the small number of prolif-erating T-cells present in biopsies were largely restricted toareas of focal tissue damage.

Statistical analysis found no correlation between glomer-ular T-cells or proliferating glomerular T-cells and serumcreatinine, creatinine clearance or proteinuria. However,both populations gave a significant correlation with glomer-ular lesions (Table 1). Interstitial T-cells and proliferatinginterstitial T-cells significantly correlated with serum creat-inine, creatinine clearance and tubulointerstitial lesions(Table 1).

DISCUSSION

This study has identified local macrophage proliferationin human GN. The role of macrophage proliferation inrenal injury and comparisons with studies of experimentalGN are discussed below.

The degree of macrophage proliferation correlated withthe severity of renal injury. This was evident by the smallnumbers of proliferating macrophages seen in less aggres-sive forms of GN compared to that present in aggressiveforms of disease. In addition, macrophage proliferationgave a highly significant correlation with the loss of renalfunction and histologic damage. Similar findings have beenmade in animal studies. Few proliferating macrophages areseen in mild forms of renal injury, such as anti-Thy-1nephritis, immune complex GN and lipid-induced glomer-ular injury [17–20]. In contrast, prominent macrophageproliferation have been demonstrated in aggressive ratmodels of allograft rejection and crescentic GN [15, 16].

The high level of macrophage proliferation seen in

Table 1. Correlation of macrophage and T-cell proliferation with renal function and histologic damage

SCrmmol/liter

CCrml/min

Proteinuriamg/24 hr

Glom hyp(0-3)

Glom seg les Crescents TI lesions

%

Glomerulus cells/gcsKP11 macrophages 0.60d 20.56d 0.04 0.65d 0.67d 0.60d —KP11 PCNA1 cells 0.60d 20.52d 20.03 0.62d 0.64d 0.55c —UCHL11 T-cells 0.11 20.22 20.06 20.07 20.17 20.05 —UCHL11 PCNA1 cells 0.03 20.13 20.02 20.12 20.13 0.02 —

Interstitium cells/mm2

KP11 macrophages 0.66d 20.64d 0.03 — — — 0.75d

KP11 PCNA1 cells 0.69d 20.58d 20.02 — — — 0.66d

UCHL11 T-cells 0.26 20.41 0.06 — — — 0.70d

UCHL11 PCNA1 cells 0.29a 20.33b 0.02 — — — 0.69d

The number of macrophages (KP11 cells), proliferating macrophages (KP11 PCNA1 cells), T-cells (UCHL11 cells) and proliferating T-cells(UCHL11 PCNA1 cells) in both the glomerulus and interstitium was compared with parameters of renal function (Pearson correlation coefficient) andhistologic damage (Spearman’s correlation coefficient). Abbreviations are: SCr, serum creatinine; CCr, creatinine clearance; Glom hyp, glomerularhypercellularity; Glom seg les, glomerular segmental proliferaive lesion; Tub lesions, tubulointerstitial lesions. Data are from 75 patients from all diseasecategories. a P , 0.05, b P , 0.01, c P , 0.001, d P , 0.0001

Yang et al: Macrophage proliferation in human GN148

aggressive forms of human GN suggests that local prolifer-ation is an important mechanism of macrophage accumu-lation within the injured kidney. However, there must be arapid turnover of inflammatory macrophages to prevent thekidney from simply filling up with these cells. This issue hasbeen explored in temporal studies of rat crescentic GN.Local proliferation is a major mechanism of macrophageaccumulation during disease development, while apoptosis

and trafficking to the draining lymph node are the mainroutes by which macrophages are rapidly removed from thekidney [21, 28, 29].

A striking feature of local macrophage proliferation inhuman GN is that it is largely restricted to focal areas oftissue damage. The high level of proliferation is a mecha-nism of amplifying macrophage numbers within these le-sions and thus increasing the potential for macrophage-mediated renal injury. Local macrophage proliferation was

Fig. 4. Quantitation of macrophage accumulation and macrophage pro-liferation in human glomerulonephritis (GN). The number of KP11macrophages (M) and KP11PCNA1 proliferating macrophages (f) wasscored in glomeruli (A) and the interstitium (B). Numbers in parenthesesindicate the percentage of KP11 cells expressing PCNA. Data are shownas mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 compared to normalby Mann Whitney U-test.

Fig. 5. Quantitation of T-cell accumulation and T-cell proliferation inhuman glomerulonephritis (GN). The number of UCHL11 T-cells (M)and UCHL1PCNA1 proliferating T-cells (f) was scored in glomeruli (A)and the interstitium (B). Numbers in parentheses indicate the percentageof UCHL11 cells expressing PCNA. Note that no UCHL11PCNA1 cellswere observed in glomeruli or the interstitium of normal kidney. Data areshown as mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 compared tonormal by Mann Whitney U-test.

Yang et al: Macrophage proliferation in human GN 149

prominent in glomerular segmental proliferative lesions,crescents, tubulointerstitial lesions and multinucleated gi-ant cell formation within granulomatous-like lesions inhuman GN. Similar observations have been made in ratcrescentic GN [21, 30]. In particular, macrophage prolifer-ation is thought to precede cell fusion in the formation ofmacrophage multinucleated giant cells [31].

What is the mechanism driving local macrophage prolif-eration in human GN? There is evidence to support a rolefor macrophage colony-stimulating factor (M-CSF), a wellcharacterized macrophage growth factor, in this process.Studies of murine lupus nephritis and rat crescentic GNhave shown increased glomerular and tubular M-CSFmRNA and protein expression that correlates with macro-phage proliferation and accumulation [32–34]. In addition,a recent study of human GN found an increase in glomer-ular M-CSF protein expression in cases of IgA nephropathyand lupus nephritis that correlated with glomerular prolif-eration and macrophage infiltration [35]. The potentialinvolvement of other cytokines in local macrophage prolif-eration, such as macrophage migration inhibitory factor(MIF), IL-13, granulocyte-macrophage colony-stimulatingfactor (GM-CSF) and macrophage stimulating protein,have yet to be examined in human or experimental glomer-ulonephritis.

Evidence for T-cell proliferation in human GN was alsoshown in this study, although at much lower levels com-pared to macrophage proliferation. This is consistent withprevious studies demonstrating T-cell activation in humancrescentic GN on the basis of expression of the interleu-kin-2 receptor [36]. Interstitial proliferating T-cells gave ahighly significant correlation with loss of renal function andhistologic damage, consistent with previous studies [1–4].In vitro, activated T-cells are a rich source of cytokines andgrowth factors, such as MIF, macrophage inflammatoryprotein-1b, M-CSF and GM-CSF, which have the potentialto induce blood monocyte accumulation and local macro-phage proliferation. However, further studies are neededto determine whether activated T-cells actually synthesizethese molecules within the diseased kidney, and if theyparticipate in macrophage infiltration and proliferation.

In summary, this study demonstrates that macrophageproliferation is a feature of the more aggressive forms ofhuman GN and that this local proliferation may be animportant mechanism for amplifying macrophage-medi-ated renal injury. The degree of local macrophage prolif-eration may be a useful diagnostic and prognostic indicatorfor human GN. Furthermore, the mechanisms driving localmacrophage proliferation could provide new therapeutictargets for the treatment of aggressive forms of GN.

ACKNOWLEDGMENTS

This study was part of a Renal Sister Centre programme establishedunder the auspices of the International Society of Nephrology. This studywas funded by grants from the Ghangdong Science and Technology

Council, People’s Republic of China, the Australian Kidney Foundation(#G1/97) and the National Health and Medical Research Counsil ofAustralia (#981122). We are grateful for the technical assistance of PaulCrammer of the Department of Anatomical Pathology, Monash MedicalCentre. The publication of Figure 3 in color was made possible by a grantfrom Janssen Cilag Pty Ltd.

Reprint requests to Dr. Hui Y. Lan, Department of Nephrology, MonashMedical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia.E-mail: huiLan@its-mmcc1.cc.monash.edu.au

APPENDIX

Abbreviations used in this article are: APAAP, alkaline phosphataseanti-alkaline phosphatase complexes; cres GN, crescent glomerulonephri-tis; FCS, fetal calf serum; FGS, focal glomerulosclerosis; GBM, glomer-ular basement membrane; gcs, glomerular cross section; GM-CSF, gran-ulocyte-macrophage colony-stimulating factor; GN, glomerulonephritis;IgAN, immunoglobulin A nephropathy; lupus II, lupus nephritis WHOclass II; lupus IV, lupus nephritis WHO class IV; mAb, monoclonalantibodies; MCD, minimal change disease; MCPGN, mesangiocapillaryproliferative glomerulonephritis; M-CSF, macrophage colony-stimulatingfactor; memb GN, membranous GN; MIF, macrophage migration inhib-itory factor; non-IgA MPGN, non-IgA mesangial proliferative glomerulo-nephritis; PAP, peroxidase anti-peroxidase complexes; PBS, phosphatebuffered saline; PCNA, proliferating cell nuclear antigen; vasc GN,nephritis associated with vasculitis.

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