Increased mRNA expression of metalloproteinase-9 in peripheral blood monocytes from patients with immunoglobulin a nephropathy

Increased mRNA Expression of Metalloproteinase-9 in Peripheral Blood Monocytes From Patients With Immunoglobulin A Nephropathy Hikaru Koide, MD, Tsukasa Nakamura,

MD, lsao Ebihara, MD, and Yasuhiko Tomino, MD

0 We examined metalloproteinase (MMP)-1, -2, -3, and -9 mRNA expression by peripheral blood monocytes from 50 patients with immunoglobulin A (IgA) nephropathy, 20 with membranous nephropathy, 10 with minimal-change nephrotic syndrome, five with focal glomerulosclerosis, 30 with non-IgA proliferative glomerulonephritis, and 40 healthy normal controls who were comparable with regard to age and sex. Monocytes from patients with IgA nephropathy expressed a higher level of MMP-9 mRNA than those from patients with other forms of glomerulonephritis or from healthy controls (MMP-9 to glyceraldehyde-3-phosphate dehydrogenase ratio: IgA nephropathy, 1.68 k 0.24; membranous nephropathy, 0.22 i 0.08; minimal-change nephrotic syndrome, 0.24 i 0.06; focal glomerulosclerosis, 0.32 i 0.08; non-IgA proliferative glomerulonephritis, 0.30 k 0.12; and healthy controls, 0.16 i 0.04). When the biopsy specimens were classified into four grades according to the severity of glomerular and interstitial pathology, highly significant differences were observed among MMP-9 mRNA levels in monocytes from all four groups of patients with IgA nephropathy (grade I, 0.44 i 0.09; grade II, 1.06 i- 0.26; grade Ill, 2.22 ? 0.68; grade IV, 2.86 ? 0.88). In addition, MMP-9 mRNA levels from patients with IgA nephropathy correlated with urinary protein excretion (P < 0.001). However, we detected minimal mRNA expression of MMP-1, -2, and -3 by peripheral blood monocytes from patients with IgA nephropathy or other forms of glomerulonephritis and from normal healthy controls. Our results suggest that increased MMP-9 mRNA expression in circulating monocytes may contribute to the progression of IgA nephropathy. 0 1996 by the National Kidney Foundation, Inc. INDEX

WORDS:

Glomerulonephritis;

metalloproteinase;

extracellular

E

VOLUTION of inflammatory and immune reactions is dependent on the recruitment and migration of circulating leukocytes to sites of injury or antigen deposition.’ Immunoglobulin A (IgA) nephropathy is reported more frequently in the Asian population, and is regarded as the most common form of glomerulonephritis and a major cause of end-stage renal disease.* Some investigators have reported that either lymphocytes or polymorphonuclear leukocytes are activated in IgA nephropathy.3-5 Arima et aI6 suggested that monocytes may play an important role in the pathogenesis of mesangial hypercellularity, irreversible glomerular damage, and interstitial tissue injury in IgA nephropathy. A modest influx of monocytes and/or macrophages is From the Division of Nephrology, Department of Medicine, Juntendo University School of Medicine, Koto Hospital, Tokyo, Japan. ReceivedAugust 24, 1995; accepted in revisedform February 6, 1996. Supported in part by a Grant-in-Aid for Intractable Diseases from the Ministry of Health and Welfare of Japan. Address reprint requests to Hikaru Koide, MD, Division of Nephrology, Department of Medicine, Juntendo University School of Medicine, Koto Hospital, 6-8-5 Ojima, Koto-ku, Tokyo 136, Japan. 0 1996 by the National Kidney Foundation, Inc. 0272-6386/96/2801-0004$3.00/O

32

American

Journal

matrix;

disease

activity.

sometimes demonstrable by immunohistochemistry in patients with focal-segmental changes in IgA nephropathy.7,8 However, little is known about the association between the activation of peripheral blood monocytes and renal injuries in IgA nephropathy. Human monocytes have been demonstrated to secrete four metalloproteinases (MMPs), including MMP-1, -2, -3, and -9.’ The activated monocytes may participate in extracellular matrix turnover. We previously reported that mRNA levels of MMPs are abnormally regulated in the renal tissues of glomerulonephritis in animal models.lO~ll The complete amino acid sequence of MMP9 has been deduced from cDNA sequencing. It shares homology with tissue MMP-1 and other members of the MMP family.12 Metalloproteinase-9 may be associated with cellular migration, invasion, activation, and catabolism under certain pathologic conditions.‘3 Metalloproteinase-9 is prominently produced and secreted by blood monocytes/tissue macrophages, indicating that MMP-9 plays an important role in inflammatory processes.14 Persistence of an inflammatory stimulus favors excessive recruitment of inflammatory cells with the potential for leukocyte-mediated tissue damages as the basis of chronic inflammatory diseases, including IgA nephropa-

of Kidney

Diseases,

Vol 28, No 1 (July),

1996:

pp 32-39

MMP GENE EXPRESSION

IN GLOMERULONEPHRITIS

thy.’ However, gene regulation of MMPs in peripheral blood monocytes from patients with glomerulonephritis remains to be defined. The purpose of the present study was to determine whether mRNA levels of MMPs in monocytes were altered in IgA nephropathy or other types of glomerulonephritis. We also examined the relationship between MMP gene expression by monocytes and renal histopathology or urinary protein excretion in IgA nephropathy. MATERIALS

AND

METHODS

Patients Between January 1992 and December 1994 we recruited 30 male and 20 female patients with IgA nephropathy (aged 16 to 60 years; mean age, 33.4 years); 65 patients with other types of glomerulonephritis (aged 18 to 64 years; mean age, 35.2 years), including 20 with membranous nephropathy, 10 with minimal-change nephrotic syndrome, five with focal glomerulosclerosis, and 30 with proliferative glomendonephritis without IgA deposition; and 25 male and 15 female age-matched healthy controls (aged 18 to 58 years; mean age, 34.2 years). The diagnosis of IgA nephropathy was based on renal biopsy findings, including the predominance of IgA deposition with or without C3 staining in the glomerttlar mesangial areas by immunofluorescence. None of the patients had any clinical or laboratory evidence of liver disease, Henoch-Schiinlein purpura, or other systemic diseases. All patients had been free from infection or macroscopic hematuria for at least 4 weeks before the initial investigation. The histologic changes in renal tissues obtained from patients with IgA nephropathy were classified as described previously.‘5 Each specimen examined by light microscopy contained at least eight glomeruli. Among the 50 patients with IgA nephropathy, 12 were classified as histologic grade I, 18 as grade II, 15 as grade II, and five as grade IV. Proteinuria was used as an indicator of disease activity. The patients were judged to have clinically active disease when the 24hour urinary protein excretion exceeded 1.0 g. Patients were not receiving treatment at the time of renal biopsy. Patients were excluded if serum creatinine was over 2.0 mg/dL, endogenous creatinine clearance was lower than 60 mL/min, or blood pressure was higher than 180/100 mm Hg to avoid the effect of chronic renal failure or hypertension. The conditions were previously described.16 We evaluated the clinical course of 20 patients with IgA nephropathy during hospitalization. We analyzed mRNA levels of peripheral blood monocytes from patients with IgA nephropathy before treatments and 1 and 3 months after treatments.

Preparation

of Peripheral

Blood Monocytes

Peripheral blood mononuclear cells were obtained by density gradient centrifugation and fractionation of 50 mL heparinized blood. The monocyte preparation was over 95% pure, as judged by nonspecific esterase staining, and more than

33 98% of cells were viable.16 Approximately were stored in liquid nitrogen for further

Extraction and Quantitation

3 x lo6 monocytes studies.

of Cellular RNA

Total cellular RNA was extracted using vanadylribonucleoside complexes.‘6 The total RNA concentration was determined spectrophotometrically at 260 nm. The purity of the RNA preparation was assessed by measuring the optical density ratio of 260 to 280 nm. RNA samples (1 pg) were denatured and electrophoresed through 0.7% agarose gels containing 2.2 mol/L formaldehyde and transferred by capillary blotting onto nylon filters (GeneScreen; New England Nuclear, Boston, MA). Before transferring RNA onto the nylon membrane, the ethidium-stained gels were visualized under ultraviolet illumination to determine the position of 28s and 18s ribosomal RNA bands, to assess the integrity of RNA, and to verify that equal amounts of RNA were loaded. RNA was fixed to the nylon membrane by ultraviolet irradiation for 5 minutes at 254 nm. Hybridization was performed in 1% bovine serum albumin, 7% sodium dodecylsulfate, 0.5 mol/L NaHZP04, 1 mmol/L EDTA, and 100 pg/mL sonicated salmon sperm DNA for 20 hours at 65’C.17 The filters were washed three times in 0.5% bovine serum albumin, 5% sodium dodecylsulfate, 40 mmol/L NaH,P04, and 1 mmol/L EDTA at 65°C followed by three washes in 1% sodium dodecylsulfate, 40 mmol/L NaHZP04, and 1 mmol/L EDTA at 65”C.‘7 The filters were then exposed to Kodak X-OMATAR film at -70°C with the aid of intensifying screens. We checked mRNA levels from monocytes in the same patient on several occasions before treatments (three times), and found that the levels of mRNA are consistent.

Description

of cDNA Probes

Human MMP-1 (H5A) and MMP-3 (H3A) cDNA probes were kindly provided by Dr Markku Kurkinen (University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ) and Dr Hideaki Nagase (University of Kansas Medical Center, Kansas City, KS).“.r9 cDNA probes for human MMP-2 and MMP-9 were kindly provided by Drs Gregory I. Goldberg and Barry L. Marmer (Washington University School of Medicine, St Louis, MO).r’-” The human glyceraldehyde-3-phosphate dehydrodrogenase (GAPDH) cDNA was obtained from the American Type Culture Collection (Rockville, MD). The cDNA inserts were excised and labeled using a random-primed cDNA labeling kit (Boehringer-Mannheim-Yamanouchi K.K., Tokyo, Japan) with 3*P-labeled deoxynucleotides (New England Nuclear, Boston, MA) to a specific activity of lo9 cpm/pg.”

Statistical Analysis The quantitative densitometric measurements on the Northem blots were normalized to the signal for GAPDH mRNA. The ratio of the absorbance areas of each mRNA signal divided by the absorbance area of the GAPDH mRNA signal were reported as mean values 5 SEM. We used regression analysis to examine the relationship between MMP-9 mRNA and grade of disease and proteinuria. In addition, we evalu-

KOIDE

ET AL

* P
membranous nephropathy (n=20)

ated MMP-9 mRNA unpaired t-test.

* --CL

and proteinuria

minimal change focal nephrotic syndrome glomerulosclerosis (n=l 0) (n.5)

before

treatments

rL

*

non-IgA normal healthy proliferative controls glomerulonephritis (n=40) (n-30)

Fig 1. Transcription of mRNA for MMP-9 in peripheral blood monocytes. The monocytes purified from patients with IgA nephropathy (n = 50) expressed more MMP-9 mRNA than those from healthy controls (n = 40) or from patients with other types of glomerulonephritis (n = 65). Data are expressed as mean values i SEM.

by

RESULTS The MMP-9 mRNA levels in peripheral blood monocytes expressed as a ratio of MMP-9 to GAPDH mRNA are illustrated in Fig 1. The MMP-9 gene expression in monocytes isolated from patients with IgA nephropathy (1.68 + 0.24) was significantly higher than that of healthy controls (0.16 2 0.04; P < 0.001) or of patients with other types of glomerulonephritis (membranous nephropathy, 0.22 + 0.08, P < 0.001; minimal-change nephrotic syndrome, 0.24 2 0.06, P < 0.001; focal glomerulosclerosis, 0.32 + 0.08, P < 0.001; and non-IgA proliferative glomerulonephritis, 0.30 k 0.12, P < 0.001). When the patients with IgA nephropathy were stratified according to the severity of their glomerular and interstitial lesions, an apparent increase in MMP-9 gene expression in peripheral blood monocytes was associated with an increase in histopathologic grading (r = 0.784; P < 0.001) (Fig 2). Grade I or II patients had low MMP-9 mRNA levels, whereas those with grade III or IV expressed high levels. Metalloproteinase-9 mRNA levels and proteinuria were highly correlated (r = 0.832, P < O.OOl), with significantly higher MMP-9 mRNA levels in

. .

. l *

.

*

. .

. .

I (rl=12)

II

III

(k18)

(k15)

Histopathological grading Fig 2. Relationship 9 mRNA in peripheral ologic grading in MMP-9 mRNA in with the severity nephropathy. r = indicate the mean.

iv (n=5)

(Grade)

between transcription of MMPblood monocytes and histopathIgA nephropathy. The increase in monocytes correlated significantly of histopathologic grading for IgA 0.764; P < 0.001. Horizontal bars

MMP

GENE

EXPRESSION

35

IN GLOMERULONEPHRITIS

b----IgA

Fig 3. Northern blot analysis of mRNA for MMP-9 (A) and GAPDH (B) in the RNA extracts of peripheral blood monocytes from patients with IgA nephropathy (histologic grade IV), other types of glomerulonephritis, and normal controls. Total cellular RNA (2 pg/lane) was extracted, electrophoresed through 0.7% agarose gel containing formaldehyde, and transferred to GeneScreen filters. Lanes 1 to 5, IgA nephropathy; lanes 6 to 10, other types of glomerulonephritis; and lanes 11 to 15, normal healthy controls.

nephropathy

12345

Other types of glom~ruloneph~~is 678

Normal healthy controls I;12

i ‘jll

IgA nephropathy

those with active proteinuria (> 1.O g/d) than in those with inactive proteinuria (< 1.0 g/d) (P < 0.001). However, we did not observe any correlation with MMP-9 mRNA expression and urinary protein excretion in patients with other types of glomerulonephritis. Figure 3 illustrated the Northern blot analysis of mRNA for MMP-9 and GAPDH from peripheral blood monocytes in patients with IgA nephropathy, other types of glomerulonephritis, and normal healthy controls. A single MMP-9 mRNA transcript of 3.3 Kb was detected in the peripheral blood monocytes. In contrast, we detected minimal MMP-1, -2, and -3 mRNA expression from peripheral blood monocytes in patients with IgA nephropathy, other types of glomerulonephritis, and normal healthy controls. Furthermore, we evaluated the clinical course of 20 patients during hospitalization. The treatment regimen in patients with IgA nephropathy and the MMP-9 to GAPDH mRNA ratio is shown in Table 1. These data were shown before treatments and 3 month after treatments. The MMP-9 to GAPDH mRNA ratio in the peripheral blood monocytes of eight patients with IgA nephropathy decreased gradually, as did urinary protein excretion, after treatment with prednisolone. However, ratios of the other 12 patients were not altered by treatment with other drugs, including dipyridamole, heparin, cilostazol, or dilazep dihydrochloride. Figure 4 shows the

Other types of glomerulonephritis

MMP-9

Normal healthy controls

Northern blot analysis of mRNA for MMP-9 from peripheral blood monocytes in five patients with IgA nephropathy (histologic grade IV) before or 3 months after treatments. DISCUSSION

Recent studies have suggested that the immunoregulatory function of T lymphocytes is related to disease activity in patients with IgA nephropathy.22,23We report that MMP-9 gene expression is increased in the peripheral blood monocytes of patients with IgA nephropathy. Metalloproteinase-9 synthesis has been demonstrated in polymorphonuclear leukocytes, alveolar macrophages, and epidermal keratinocytes.‘2’w”25 Some investigators have reported that MMP-9 mRNA was expressed in malignant tumors.26-2xMetalloproteinase-9 mRNA also is expressed by eosinophils associated with nodular basal cell carcinoma. On the other hand, MMP-9 is thought to be primarily a product of migratory cells, and thus, its activity may be involved in various conditions associated with inflammation.9,30 Metalloproteinase-9 is a prominent secretory product of both blood monocytes and alveolar macrophages; its secretion can be upregulated by lipopolysaccharide.’ Furthermore, small amounts of this enzyme are secreted even by U937 monocyte-like cells in the basal state.’ Thus, the capacity for MMP-9 expression appears to develop at a comparative early stage of mononuclear phago-

36

KOIDE Table

1. Treatment

Regimen

in Patients

With

lmmunoglobin

MMP-SIGAPDH Patient No.

Histologic Grade

1 2 3

II II II

4 5 6 7 8 9

II Ill Ill Ill Ill Ill

10 11

Ill Ill

12 13

Ill Ill

14 15 16 17 18 19

Ill III IV IV IV IV

20

IV

Drug Dipyridamole Dipyridamole Cilostazol and dilazep, dihydrochloride Watfarin and cilostazol Dipyridamole Dipyridamole Prednisolone and cilostazol Prednisolone and dipyridamole Prednisolone, dipyridamole, and watfarin Prednisolone and dipyridamole Cilostazol and dilazep, dihydrochloride Dipyridamole and enalapril Prednisolone, dipyridamole and warfarin Prednisolone and dipyridamole Dipyridamole Dipyridamole and watfarin Dipyridamole, heparin, and warfarin Dipyridamole, heparin and watfarin Methylprednisolone, heparin, and cilostazol Prednisolone, dipyridamole, heparin, and watfarin

cyte differentiation. However, extensive studies on the gene expression of MMP-9 in peripheral blood monocytes from patients with glomerulonephritis have not been completed. Monocytes may be activated during their journey through the glomerular microvascular bed and interstitium to release MMP-9 locally. Metalloproteinase-9 secreted by peripheral blood monocytes would probably access the glomerulus, inter&Gum, or blood-vessel wall. Such a mechanism is consistent with our finding of a positive correlation between MMP-9 mRNA levels in monocytes and histopathologic changes or urinary protein excretion. We showed previously that MMP-9 mRNA is absent in renal tissues of normal control NZW mice until 48 weeks of age, whereas it is increased in NZB/W Fl mice with the progression of nephritis.” In the kidneys of NZB/W Fl mice, we showed that many mononuclear cells infiltrate as the nephritis progressed. It is probable that

Pretreatment

ET AL

A Nephropathy

mRNA Ratio Posttreatment

Proteinuria Pretreatment

(g/d) Posttreatment

1.38 1.42 1.86

1.40 1.36 1.80

1.2 1.4 1.5

0.4 0.6 0.9

2.04 1.86 1.64 3.22 3.46 2.68

1.98 1.80 1.52 1.36 1.62 1.40

1.4 1.6 1.4 2.2 3.2 2.4

0.8 0.8 0.6 0.8 0.6 0.8

2.52 1.76

1.08 1.62

3.0 1.6

0.4 1.0

1.90 3.20

1.86 1.26

1.4 3.8

0.8 0.4

2.88 1.66 2.46 2.88 1.86 3.62

1.34 1.52 2.32 2.68 1.78 1.36

4.2 0.8 1.6 1.8 2.2 3.8

0.8 0.4 1.2 1.0 1.4 0.6

3.48

1.42

3.0

0.4

one source of the enhanced MMP-9 mRNA is the monocyte/macrophage cell population. In the present study, we did not examine whether the high levels of MMP-9 mRNA are associated with a high production of protein. It would be needed to perform Western blot, nuclear run-off, and mRNA stability analyses in further studies. Matrisian showed that positive regulation of MMP gene expression occurs at the level of transcriptional activation.31 Studies on the regulation of MMP transcription have resulted in a better understanding of how cytokines and oncogenes may control gene expression. Several lines of evidence suggest that c-Fos and cJun are important regulators of MMP gene expression. In preliminary studies, we determined that MMP-9 cells shown to be positive by immunohistochemistry in renal tissues of IgA nephropathy were associated with MMP-9 mRNA levels of peripheral blood monocytes (unpublished data). The important issue of whether MMP-9

MMP

GENE

EXPRESSION

IN GLOMERULONEPHRITIS

Fig 4. Northern blot analysis of mRNA for MMP-9 in the RNA of peripheral blood monocytes from patients with IgA nephropathy (histologic grade IV) before or 3 months after treatments. (A) Patient 16, (B) patient 17, (C) patient 18, (D) patient 19, and (E) patient 20 (Table 1).

mRNA levels from peripheral blood monocytes in IgA nephropathy are associated with MMP-9 mRNA signals in renal tissues by in situ hybridization is now addressed in progression. Induction of MMP-9 in articular chondrocytes and skin fibroblasts by interleukin-1 or tumor necrosis factor-a and in human rheumatoid synovial cells by a phorbol ester also has been reported.32 Welgus et al9 reported that the treatment of human alveolar macrophages with lipopolysaccharide enhances the production of MMP-9. In addition, activated monocytes/macrophages produce interleukin-1 and tumor necrosis factor(Yalong with MMP-9. It is therefore possible that these cytokines upregulate MMP-9 mRNA in peripheral blood monocytes from patients with IgA nephropathy. Lai et a133recently demonstrated that CD4+ T cells from patients with IgA nephropathy expressed a higher level of transforming growth factor-p mRNA than that of healthy controls or other types of glomerulonephritis. We also found that peripheral blood monocytes from patients with IgA nephropathy expressed a higher level of transforming growth factor-p mRNA (unpublished data). Transforming growth factor-p may enhance MMP-9 transcripts in human monocytes and contribute to persistent leukocyte accumula-

tion.(j Some investigators reported transforming growth factor-p stimulation of monocytes to express MMP-9.‘2*20 We previously reported that enhanced expression of proto-oncogene, including c-fos and cj,,, may contribute to the progression of IgA nephropathy.34 The induction of MMP-9 may be associated with enhanced c-fos and c-jun mRNA expression in IgA nephropathy. Ogata et alI3 suggested that MMP-3 is the most likely candidate that participates in the activation of MMP-9 in cell lines. However, we detected minimal MMP3 mRNA in monocytes from patients with glomerulonephritis including IgA nephropathy. Welgus et al9 reported that MMP-3 is not secreted by human monocytes or by U937 cells in either their basal or differentiated state. We detected minimal MMP-1, MMP-2, and MMP-3 mRNA in monocytes from patients with glomerulonephritis. It is necessary to accumulate information on how MMP-9 mRNA is expressed and to obtain insights into basic mechanisms by which cytokines and oncogenes alter the gene expression pattern and subsequent renal injury in IgA nephropathy. We studied the clinical course of 20 patients with IgA nephropathy during hospitalization (> 3 months). The MMP-9 mRNA levels in patients

38

KOIDE

with IgA nephropathy decreased gradually, as did proteinuria after prednisolone treatment. However, MMP-9 mRNA levels in patients with IgA nephropathy treated with other drugs did not change throughout treatment, despite a decrease in proteinuria. Steroids have been shown to specifically diminish MMP-1 and MMP-3 mRNA in fibroblasts.35,36 However, little is known about the effects of steroids on MMP-9 mRNA expression. The low monocyte MMP-9 mRNA levels in patients with IgA nephropathy receiving prednisolone is probably secondary to immune suppression associated with deactivation of monocytes and, thereby, decreased MMP-9 mRNA expression. The mechanism by which prednisolone inhibits MMP-9 mRNA expression in peripheral blood monocytes is unknown at this time. This finding may be due to a reduction in the rate of MMP-9 gene transcription, an inhibition in the transport of mRNA from the nucleus to cytoplasm, or to the fact that specific protein synthesis is required to mediate this inhibition. In summary, we have demonstrated that patients with IgA nephropathy have peripheral blood monocytes that express MMP-9 mRNA at higher levels than in other types of glomerulonephritis and normal healthy controls. Moreover, these mRNA levels correlate with disease activity. Our results suggest another role for MMP-9 in monocytes of patients with IgA nephropathy. Thus, MMP-9 mRNA expression may be used to monitor disease activity in IgA nephropathy. ACKNOWLEDGMENT The authors thank Dr M. Kurkinen, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ, and Dr H. Nagase, University of Kansas Medical Center, Kansas City, KS, for providing the human MMP-1 and MMP-3 cDNA probes, and Drs G.I. Goldberg and B.L. Marmer, Washington University School of Medicine, St. Louis, MO, for providing human MMP-2 and MMP-9 cDNA probes. REFERENCES 1. Wahl SM, Allen JB, Weeks BS, Wong HL, Klotman PE: Transforming growth factor-p enhances integrin expression and type IV collagenase secretion in human monocytes. Proc Nat1 Acad Sci U S A 90:4577-4581, 1993 2. Emancipator SN: IgA nephropathy. Morphologic expression and pathogenesis. Am J Kidney Dis 23:451-462, 1994 3. Yano N, Miyazaki M, Endoh M, Kuramoto T, Eguchi

ET AL

K, Yagame M, Nomoto Y, Sakai H: Increase of CD23-positive cells in peripheral blood from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis. Nephron 60:404410, 1992 4. Sato M, Haizawa H, Asakura H: Relationship between serum IgA levels and CD4+ subsets in IgA nephropathy. Nephron 68:20-24, 1994 5. Kashem A, Endoh M, Nomoto Y, Sakai H, Nakayama H: FCCXRexpression on polymorphonuclear leukocyte and superoxide generation in IgA nephropathy. Kidney Int 45:868-875, 1994 6. Arima S, Nakayama M, Naito M, Sato T, Takahashi K: Significance of mononuclear phagocytes in IgA nephropathy. Kidney lnt 39:684-692, 1991 7. Li HL, Hancock WW, Hooke DH, Dowling JP, Atkins RC: Mononuclear cell activation and decreased function in IgA nephropathy with crescents. Kidney lnt 37:1552-1556, 1990 8. Nalasco FE, Cameron JS, Hartley B, Coelho A, Hildreth G, Reuben R: lntraglomerular T cells and monocytes in nephritis: Study with monoclonal antibodies. Kidney lnt 31:1160-1166, 1987 9. Welgus HG, Campbell EJ, Cury JD, Eisen AZ, Senior RM, Wilhelm SM, Goldberg Gl: Neutral metalloproteinases produced by human mononuclear phagocytes. Enzyme profile, regulation, and expression during cellular development. J Clin Invest 86:1496-1502, 1990 10. Nakamura T, Ebihara 1, Osada S, Takahashi T, Yamamoto M, Tomino Y, Koide H: Gene expression of metalloproteinases and their inhibitor in renal tissue of New Zealand black/white Fl mice. Clin Sci 85:295-301, 1993 11. Nakamura T, Fukui M, Ebihara I, Tomino Y, Koide H: Low protein diet blunts the rise in glomerular gene expression in focal glomerulosclerosis. Kidney lnt 45:1593-1605, 1994 12. Wilhelm SM, Collier IE, Marmer BL, Eisen AZ, Grant GA, Goldberg Cl: SV-40-transformed human lung fibroblasts secrete a 92-KDa type IV collagenase which is identical to that secreted by normal human macrophages. J Biol Chem 264:17213-17221, 1989 13. Ogata Y, Enghild JJ, Nagase H: Matrix metalloproteinase-3 activates the precursor for the human matrix metalloproteinase-9. J Biol Chem 267:3581-3584, 1992 14. Thompson RW, Holmes DR, Mertens RA, Liao S, Botney MD, Mecham RP, Welgus HG, Parks WC: Production and localization of 92.kilodalton gelatinase in abdominal aortic aneurysms. J Clin Invest 96:318-326, 1995 15. Lee SMK, Rao VM, Franklin WA, Schiffer MS, Aronson AJ, Spargo BH, Katy AL: IgA nephropatby; morphologic predictors of progressive renal disease. Hum Path01 13:314322, 1982 16. Nakamura T, Ebihara 1, Shirato 1, Fukui M, Tomino Y, Koide H: Endothelin-1 mRNA expression by peripheral blood monocytes in IgA nephropathy. Lancet 342: 1147-l 148, 1993 17. Church GM, Gilbert W: Genomic sequencing. Proc Nat1 Acad Sci U S A 81:1991-1995, 1984 18. Saus J, Quinones S, Otani Y, Nagase H, Harris ED Jr, Kurkinen M: The complete primary structure of human

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matrix metalloproteinase-3. J Biol Chem 263:6742-6745, 1988 19. Sato T, Ito A, Mori Y, Yamashita K, Hayakawa T, Nagase H: Hormonal regulation of collagenolysis in uterine cervical fibroblasts. Biochem .I 275:645-650, 1991 20. Collier IE, Wilhelm SM, Eisen AZ, Marmer BL, Grant GA, Seltzer JL, Kronberger A, He C, Batter EA, Goldberg GI: H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen. .I Biol Chem 263:6579-6587, 1988 21. Feinberg AP, Vogelstein B: A technique for radiolabeling restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13, 1983 22. Feehally J, Beattie TJ, Brenchley PEC, Coupes BM: Sequential study of the IgA system in relapsing IgA nephropathy. Kidney Int 30:924-931, 1986 23. Lai KN, Leung JCK, Lai FM, Tam JS: Early T-lymphocyte activation in IgA nephropathy. Soluble interleukin2 receptor production, cellular IL-2R expression and IL-2 release. J Clin Immunol 9:485-492, 1989 24. Hibbs MS, Hoidal JR, Kang AH: Expression of a metalloproteinase that degrades native type IV collagen and denatured collagens by cultured human alveolar macrophages. J Clin Invest 80:1644-1650, 1987 25. Murphy G, Ward R, Hembry RM, Reynolds JJ, Kuhn K, Tryggavason K: Characterization of gelatinase from pig polymotphonuclear leukocytes. Biochem J 258:463-472, 1989 26. Pyke C, Ralfkiaer E, Tryggavason K, Dano K: mRNA for type IV collagenases is located in stromal cells in human colon cancer. Am J Path01 142:359-365, 1993 27. Stahle-Backdahl M, Parks WC: 92-Kd gelatinase is actively expressed by eosinophils and stored by neutrophils in squamous cell carcinoma. Am J Path01 142:995-1000,1993

39 28. Soini Y, Salo T, Oikarinen A, Autio-Harmainen H: Expression of 72 kilodalton and 92 kilodalton type IV collagenase in malignant fibrous histiocytomas and dermatofibromas. Lab Invest 69:305-311, 1993 29. Stahle-Backdahl M, Sudbeck BD, Eisen AZ, Welgus HG, Parks WC: Expression of 92 KDa type IV collagenase by eosinophils associated with basal cell carcinoma. J Invest Dermatol 99:497-503, 1992 30. Hibbs MS: Expression of 92 KDa phagocyte gelatinase by inllammatory and connective tissue cells. Matrix 1:51-57, 1992 3 1. Matrisian LM: Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet 6:121-125, 1990 32. Lefebvre V, Peeters-Joris C, Vaes G: Production of gelatin-degrading matrix metalloproteinases and inhibitors by articular chondrocyte during their dedifferentiation by serial subcultures and under stimulation by interleukin-1 and tumor necrosis factor-alpha. Biochim Biophys Acta 1094:8-18, 1991 33. Lai KN, Ho RTH, Leung JCK, Lai FNM, Li KT: Increased mRNA encoding for transforming growth factorbeta in CD4+ cells from patients with IgA nephropathy. Kidney Int 46:862-868, 1994 34. Ebihara I, Nakamura T, Suzuki S, Tomino Y, Koide H: Proto-oncogene expression in peripheral blood mononuclear cells in IgA nephropathy. Kidney Int 39:946-950, 1991 35. Frisch SM, Ruley HE: Transcription from the stromelysin promoter is induced by interleukin-1 and repressed by dexamethasone. J Biol Chem 262:16300-16304, 1987 36. Clark SD, Kobayashi DK, Welgus HG: Regulation of the expression of tissue inhibitor of metalloproteinases and collagenase by retinoids and glucocorticoids in human fibroblasts. J Clin Invest 80:1280-1288, 1987