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NDT Advance Access published online on June 25, 2007
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfm391
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Establishment of a sandwich ELISA for human megsin, a kidney-specific serine protease inhibitor

Reiko Inagi1, Yuko Izuhara1, Naoto Tominaga1, Masaomi Nangaku2, Kiyoshi Kurokawa1 and Toshio Miyata1

1Institute of Medical Sciences and Division of Nephrology, Hypertension and Metabolism, Tokai University School of Medicine, Kanagawa and 2Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Tokyo, Japan

Correspondence and offprint requests to: Toshio Miyata, Institute of Medical Sciences and Division of Nephrology, Hypertension and Metabolism, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan. Email: t-miyata{at}is.icc.u-tokai.ac.jp



   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Background. We previously identified a novel serine protease inhibitor (serpin), megsin, which is predominantly expressed in the kidney. Megsin expression is up-regulated in human and experimental renal diseases associated with mesangial proliferation and expansion, suggesting that urinary megsin may be a novel diagnostic marker for some renal diseases.

Methods. We established a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for megsin and measured urinary megsin of patients with various renal diseases.

Results. Megsin ELISA specifically detected megsin but not other serpins. The detection limit was 0.04 ng/ml, which allowed detection of urinary megsin in 3.6% of healthy individuals. The antigenic epitope in the urine detected by the ELISA was confirmed as megsin protein by time-of-flight mass spectrometry. Among patients with rapidly progressive glomerulonephritis (n = 18), 55.6% were urinary megsin-positive, while 24.1% in IgA nephropathy (n = 112) and 15.1% in chronic non-IgA glomerulonephritis (n = 245) were urinary megsin-positive, respectively. Among patients with chronic renal failure due to unknown causes (n = 74), 18.9% were positive for urinary megsin. In diabetic patients with or without nephropathy (n = 1073), 12.3% were urinary megsin-positive, while positivity of urinary megsin in patients with non-renal diseases (n = 768) was equivalent (3.3%) to that of healthy individuals. Of note, when urinary megsin-positive patients with diabetic nephropathy (n = 71) were classified into four stages by their proteinuria and estimated glomerular filtration rate, urinary megsin excretion increased as the stage progressed up to stage 3A, suggesting correlation of that with mesangial expansion level. Urinary megsin decreased in the advanced stage, probably reflecting development of glomerulosclerosis.

Conclusion. We established a high-sensitive megsin ELISA, which detects urinary megsin in some patients with renal diseases and in only a few healthy subjects. Megsin ELISA may be a novel diagnostic tool for renal diseases.

Keywords: chronic kidney disease (CKD); diabetic nephropathy; proteinuria



   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Mesangial cells maintain the structural integrity of glomeruli and regulate glomerular functions. In order to understand the pathophysiology of glomerular disorders [1], we previously cloned a human mesangium-predominant gene, megsin, which is a new member of the serine protease inhibitor (serpin) superfamily [2,3]. In IgA nephropathy and diabetic nephropathy, megsin expression in glomeruli was up-regulated [4,5]. Similar up-regulation of megsin was observed in the experimental anti-Thy1 nephritis model of rats [6]. These results suggested that increased expression of megsin gene is associated with renal disorders with mesangial expansion and proliferation. To support this notion, recent studies showed association of polymorphisms of megsin gene with susceptibility of IgA nephropathy [7]. Overexpression of human megsin in mice indeed developed progressive mesangial matrix expansion with an increase in the number of mesangial cells [8] or accelerated development of diabetic nephropathy [9].

To investigate whether urinary megsin can be a novel diagnostic marker for human renal diseases, we established a sandwich enzyme-linked immunosorbent assay (ELISA) for detection of human megsin and measured urinary megsin content in normal individuals and patients with various renal diseases including IgA nephropathy and diabetic nephropathy.



   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Monoclonal antibodies to human megsin
Spleen cells obtained from male Balb/c mice, which were immunized with recombinant human megsin derived from megsin-transfected Chinese hamster ovary cells (CHO-megsin), were fused with murine myeloma cell line, SP2 cells, utilizing the Clonacell-HY hybridoma cloning kit (StemCell Technologies, Vancouver, Canada). The hybridoma cells were then selected by the ELISA specific for megsin reported previously [5] and cloned by the limiting dilution method. The mouse antibody isotyping kit (Roche, Basel, Switzerland) was used for identification of the isotype of the monoclonal antibodies.

Binding affinity of megsin monoclonal antibodies to human megsin
To assess the binding affinity of megsin antibody to human megsin, we used the Biacore system (Biacore International AB, Uppsala, Sweden). Purified CHO-megsin (10 µg) was bound on the surface of sensor chips (CM5, Biacore International AB). Then the sensor chips coupled with CHO-megsin were incubated with each purified megsin monoclonal antibody, and the binding affinity was measured.

Western blot analysis
Human, murine or rat megsin cDNA [2,6] was cloned into glutathione S-transferase (GST) gene fusion vector, pGEX (Amersham Biosciences Corp., Piscataway, NJ). The GST-fused megsins (GST-megsin), CHO-megsin mixed with various human samples or CHO-megsin complexed with plasmin [8] were separated by SDS–PAGE, transferred to a polyvinylidene difluoride membrane (Bio Rad Laboratories, Hercules, CA) and reacted with antibodies to megsin (10 µg) as a first antibody. The reaction was developed by alkaline phosphatase substrate reaction.

Human {alpha}2-antiplasmin, {alpha}1-antitrysin (Sigma-Ardrich Corp.), antithrombin III (Wako Pure Chemical Industries, Ltd, Osaka, Japan), plasminogen activator inhibitor I (PAI-1, Molecular Innovations, Inc., Southfield, MI) or maltose binding protein (MBP)-fused PAI-2 [5] were utilized as control serpins.

Sandwich ELISA for megsin
Rabbit polyclonal antibodies (1503) as a capture antibody were obtained by immunization of rabbits with CHO-megsin followed by purification of the antiserum by megsin-specific affinity column chromatography. As a detection antibody, F(ab’)2 fraction of murine monoclonal antibody (Ms12a) was conjugated with maleimide-activated horseradish peroxidase (POD, Sigma–Ardrich Corp.) [10].

A ninety-six-well microplate (Nalge Nunc International, Rochester, NY) was coated with 1503 (0.1 µl/well) at 4°C for 16 h. After washing in phosphate-buffered saline (PBS) containing 0.05% Tween-20 (washing buffer) three times, the plate was incubated with 400 µl/well of PBS containing 25% BlockAce (Dainippon Pharmaceutical Co., Ltd, Osaka, Japan) at room temperature for 1 h. The wells were then reacted with test samples (100 µl/well) at room temperature for 2 h followed by reaction with 100 µl/well of POD-conjugated Ms12a (8.9 ng/well) at room temperature for 2 h. The plate was washed in washing buffer five times after each reaction. The development was performed by utilizing tetramethylbenzidine (TMB, MOSS, Inc., Pasadena, MD, 100 µl/well) for 30 min and was stopped by addition of 2N H2SO4 (100 µl/well). The absorbance at 450 nm was measured. The level of urinary megsin was normalized by urinary creatinine measured by Clinical Analyzer 7170 (Hitachi High-Technologies Corp., Tokyo, Japan).

Human subjects
The study was approved by the Human Research Committee of Tokai University School of Medicine. We collected urine samples from healthy volunteers (n = 55) and patients with chronic glomerulonephritis (n = 357), which included 112 of IgA nephropathy, rapidly progressive glomerulonephritis (n = 18), diabetic patients with or without nephropathy (n = 1073), chronic renal failure due to unknown causes (n = 74) and non-renal disease patients without metabolic syndromes (n = 768), who visited the out-patient clinic of Tokai University Hospital, Nagasaki University Hospital, Fujita Health University Hospital, Nagoya City University Hospital, Aichi Medical Collage Hospital and Iwata City Hospital of Japan between 2003 and 2004, with informed consent. We also collected plasma from healthy individuals or patients with renal diseases.

The diabetic patients were divided into four groups [stage 1, no microalbuminuria with normal renal function (glomerular filtration rate: GFR ≥60 ml/min); stage 2, microalbuminuria with normal renal function; stage 3A, overt proteinuria (0.5≤ urinary protein <1 g/day) with normal renal function; stage 3B, overt proteinuria (≥1 g/day) with deterioration of renal function (GFR <60 ml/min); stage 4, overt proteinuria (≥1 g/day) with deterioration of renal function (GFR <30 ml/min)] the according to the classification by the Japanese Society of Nephrology, Japanese Diabetic Society and Diabetic Nephropathy Committee. Stage 5, end-stage renal failure, was excluded in this study. GFR was estimated from serum creatinine using the MDRD study equation [11].

All urine samples were stored at –80°C until use. For the detection of urinary megsin, the urine was thawed at room temperature, vortexed with a sample diluent (50 mM Tris–HCl, pH 7.0, 0.15 M NaCl, 10% BlockAce and 0.05% Tween-20, 1:4 dilution) and subjected to ELISA.

Urinary ß2-microglobulin in some patients was also measured by radioimmunoassay.

TOF-mass spectrometry
Concentrated urine from healthy individuals was separated by SDS–PAGE. The proteins extracted from a band corresponding to megsin at the molecular weight level was digested by trypsin (Wako) and analysed by time-of-flight (TOF)-mass spectrometry (Applied Biosystems, Foster, CA) after being spotted on a MALDI plate and co-crystallized with 2,5-dihydroxybenzoic acid (DHB). Mass spectra were recorded on a MALDI-TOF instrument (oMALDI-Qq-TOF MS/MS, Applied Biosystems) at Hitachi Science Systems (Ibaraki, Japan) [12].

Statistic analysis
Data were expressed as mean ± SE. Mann–Whitney U-test and Fisher's exact test for independence, utilizing an SPSS 13.0 software, were performed to evaluate the statistical significance of various differences and the difference in the positive rate for urinary megsin between patients with each renal disease and healthy individuals, respectively. Values are considered significant at P < 0.05.



   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Establishment of monoclonal antibody to human megsin, Ms12a
We raised 20 monoclonal antibodies to CHO-megsin. Although the majority of them showed cross-reactivity with other serpins, e.g. {alpha}2-antiplasmin, {alpha}1-antitrypsin, antithrombin III, PAI-1 and MBP-fused PAI-2 by western blot analysis (data not shown), one clone, termed Ms12a, specifically reacted with CHO-megsin but not other serpins (Figure 1A). MS12a recognized another human recombinant megsin, GST-megsin, but not rat or murine megsin, demonstrating its specificity to human megsin (Figure 1B). Megsin binds to plasmin, a serine protease, and forms a complex [8]. This complex was also detected by Ms12a (Figure 1C).


Figure 1
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  		Fig. 1. Characterization of Ms12a, one of the monoclonal antibodies to human megsin derived from megsin-transfected CHO cells (CHO-megsin). (A) The cross-reactivity of Ms12a to various serpins. Ms12a specifically reacts with megsin, but not other serpins. Lane 1, {alpha}2-antiplasmin; lane 2, {alpha}1-antitrypsin; lane 3, antithrombin III; lane 4, PAI-1; lane 5, MBP-fused PAI-2; lane 6, MBP; lane 7, CHO-megsin. Lanes 1–6, 40 ng/lane; lane 7, 5 ng/lane. (B) The reactivity of Ms12a with megsin of various species. Ms12a reacts with neither rat- nor murine-derived megsin, while it specifically reacts with human megsin. Lane 1, GST-fused human megsin; lane 2, GST-fused rat megsin; lane 3, GST-fused murine megsin; lane 4, CHO-megsin. Lanes 1–3, 40 ng/lane; lane 4, 5 ng/lane. (C) The reactivity of Ms12a to megsin-plasmin complex. Ms12a reacts with not only megsin but also megsin complexed with plasmin. Lane 1, plasmin; lane 2, megsin; lane 3, megsin plus plasmin.

 
We then assessed if Ms12a cross-reacts with the constituents of human plasma or urine. Western blot analyses did not show any non-specific reaction of Ms12a with constituents of human plasma or urine derived from healthy individuals or patients with renal diseases (Figure 2, odd-numbered lanes). Importantly, Ms12a could detect megsin added in the plasma or urine, demonstrating that no constituent of plasma or urine affected the reactivity of Ms12a with megsin (Figure 2, even-numbered lanes). The isotype of Ms12a was IgG1.


Figure 2
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  		Fig. 2. Characterization of Ms12a in the presence of the constituents of human plasma (A) or urine (B). On western blot analysis, Ms12a can detect megsin added in the plasma or urine, and its reactivity was not affected by any constituents of the plasma or urine. (A) Lanes 1 and 2, the plasma (0.15 µl) of healthy individual A; lanes 3 and 4, the plasma (0.15 µl) of healthy individual B; lanes 5 and 6, the plasma (0.15 µl) of a patient with chronic non-IgA glomerulonephritis; lanes 7 and 8, the plasma (0.15 µl) of a patient with diabetic nephropathy; lane 9 CHO-megsin. (B) Lanes 1 and 2, the urine (10 µl) of healthy individual A; lanes 3 and 4, the urine (10 µl) of healthy individual B; lanes 5 and 6, the concentrated urine containing 10 µg proteins of healthy individual C; lanes 7 and 8, the concentrated urine containing 10 µg proteins of healthy individual D; lanes 9 and 10, the urine (10 µl) of a patient with chronic non-IgA glomerulonephritis; lanes 11 and 12, the urine (10 µl) of a patient with diabetic nephropathy; lane 13, CHO-megsin.

 
Establishment of a sandwich ELISA for human megsin
To establish a sandwich ELISA for human megsin, we raised three rabbit polyclonal antibodies to CHO-megsin as a capture antibody. We selected one polyclonal antibody, 1503, which shows high binding affinity in combination with Ms12a as a detection antibody by the Biacore system. The binding affinity of megsin utilizing 1503 and Ms12a is a 99.8 resonance unit (RU), while that utilizing normal rabbit IgG instead of 1503 is <15.0 RU.

Western blot analysis revealed that 1503 cross-reacts with some constituents of human plasma and urine including transferrin. However, the reactivity of 1503 with megsin did not change in the presence of plasma or urine (Figure 3).


Figure 3
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  		Fig. 3. The reactivity of 1503, a capture antibody to megsin, in the presence of human plasma (A) or urine samples (B). The reactivity of 1503 to megsin did not change in the presence of human plasma or urine. (A) Lanes 1 and 2, the plasma (0.15 µl) of healthy individual A; lanes 3 and 4, the plasma (0.15 µl) of healthy individual B; lanes 5 and 6, the plasma (0.15 µl) of a patient with chronic non-IgA glomerulonephritis; lanes 7 and 8, the plasma (0.15 µl) of a patient with diabetic nephropathy; lane 9 CHO-megsin. (B) Lanes 1 and 2, the urine (10 µl) of healthy individual A; lanes 3 and 4, the urine (10 µl) of a patient with chronic non-IgA glomerulonephritis; lanes 5 and 6, the urine (10 µl) of a patient with diabetic nephropathy; lane 7 CHO-megsin.

 
We established a sandwich ELISA system utilizing megsin polyclonal antibody, 1503, as a capture antibody and megsin monoclonal antibody, Ms12a, and evaluated its specificity and sensitivity by measuring various concentrations of CHO-megsin. The suitable detection range and the detection limit of CHO-megsin-added normal urine by this ELISA system was between 0.078 and 5.0 ng/ml (R2 = 0.995) and 0.04 ng/ml, respectively (Figure 4). When CHO-megsin was added in urine and pre-treated with Ms12a, megsin was not detected by the ELISA system (data not shown). In addition, the constituents of plasma or urine including transferrin, urea, albumin, IgG, IgA, IgM, ascorbate, glucose, haemoglobin or bilirubin did not affect detection of megsin by this sandwich ELISA (data not shown). The change of pH of test samples also did not influence sensitivity of the the ELISA (Figure 5).


Figure 4
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  		Fig. 4. The calibration curve of urinary megsin ELISA. The sensitivity of the sandwich ELISA system was assessed by measuring various concentrations of recombinant CHO-megsin. The suitable detection range and the detection limit of CHO-megsin-added normal urine by this system were between 0.078 and 5.0 ng/ml (R2 = 0.995) and 0.04 ng/ml, respectively.

 

Figure 5
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  		Fig. 5. Influence of pH on the sandwich ELISA for human megsin. To assess the influence of pH on megsin ELISA system, various concentrations of CHO-megsin with different pH were measured. The change of pH of test samples’ dose not influence the sensitivity of this system. Closed circles, 5 ng/ml; open circles, 2.5 ng/ml; closed triangles, 1.25 ng/ml; open triangles, 0.31 ng/ml. PBS, phosphate-buffered saline; TBS, Tris-buffered saline.

 
Evaluation of sensitivity and specificity of the novel sandwich ELISA for human megsin
We then tried to measure urinary megsin of human subjects utilizing the novel sandwich ELISA. We defined urinary megsin concentration of higher than the detection limit (0.04 ng/ml) as positive. Among 55 healthy individuals, two samples were positive (3.6%) for megsin (0.19 and 0.54 ng/gCr) (Figure 6).


Figure 6
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  		Fig. 6. Urinary megsin levels in various kidney diseases. The amounts of urinary megsin of 2290 patients with or without renal diseases, and 55 normal individuals were measured by the sandwich ELISA in triplicate and normalized by urinary creatinine. The urine samples were diluted (1: 4) with a sample diluent. We defined urinary megsin concentration of >0.04 ng/ml (detection limit) as positive. The percentage of urinary megsin-positive subjects in patients with renal diseases was markedly higher than that in patients without renal diseases or healthy subjects. CRF, chronic renal failure.

 
To confirm the existence of megsin in urine, we highly concentrated (1000-fold) urine from healthy subjects, who are positive for megsin as shown in Figure 6. The protein, which corresponded to megsin at the molecular weight level by SDS–PAGE, was identified as megsin by TOF-mass spectrometry (Figure 7). Therefore, we conclude that our megsin ELISA indeed detected megsin excreted into urine.


Figure 7
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  		Fig. 7. Confirmation of urinary megsin excretion by TOF-mass spectrometry. The 1000-fold-concentrated urine from healthy individuals, whose urinary megsin were positive by the sandwich ELISA as shown in Figure 6, was separated by SDS–PAGE. A protein with a molecular mass of ~45 kDa, corresponding to megsin by western blot analysis utilizing Ms12a, was digested by trypsin and analysed by TOF-mass spectrometry. (A) The spectrum of TOF-mass spectrometry of the 45-kDa protein, corresponding to megsin, digested by trypsin. (B) Amino acid sequence of human megsin (database accession No. AAC64506). The amino acid sequence matched with that of molecule identified by TOF-mass spectrometry was shown in red bold.

 
Urinary megsin was detected in the patients with various renal diseases
We then measured the levels of urinary megsin in 2290 patients with or without various renal diseases. Range of the percentage of positivity for urinary megsin from the patients with various renal diseases was between 15.1 and 55.6% and their positive ratio was significantly higher than that in healthy individuals or patients with non-renal diseases (3.3%, P < 0.05) (Figure 6). Diabetic patients with or without nephropathy also showed a tendency of an increased megsin-positive percentage (12.3%) as compared with that in healthy individuals (P = 0.052).

To evaluate the possibility that increased urinary megsin excretion was due to the disturbance of tubular re-absorption, we analysed the correlation between urinary megsin level and tubular re-absorption damage. In urinary megsin-positive patients (n = 37) with various renal diseases, the megsin excretion level did not correlate with urinary ß2-microglobulin level, an indicator of tubular re-absorption dysfunction (R2 = 0.1329, Figure 8).


Figure 8
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  		Fig. 8. The correlation between urinary megsin level and tubular re-absorption dysfunction. In 37 patients with various kidney diseases, who were positive for urinary megsin, urinary ß2-microglobulin, a marker for tubular re-absorption dysfunction, was measured. The amount of megsin excretion did not correlate with that of ß2-microglobulin, suggesting that megsin excretion level did not correlate with tubular re-absorption dysfunction.

 
Urinary megsin excretion increased as the stage progressed in the patients with diabetic nephropathy
Our previous in situ hybridization studies demonstrated up-regulation of megsin expression in the mesangial area of biopsy samples obtained from patients with diabetic nephropathy [4]. We therefore investigated the urinary megsin content in relation to the stage of diabetic nephropathy. Diabetic patients (n = 71) with nephropathy who were positive for urinary megsin and had the clinical record of proteinuria and estimated GFR, were divided into four stages by their urinary protein levels and GFR (Figure 9). Up to stage 3A, urinary megsin excretion increased as the stage progressed (stages 1 vs 3A, P < 0.01, Figure 9). Of note, after stage 3B, urinary megsin levels decreased and did not correlate with the degree of proteinuria or deterioration of estimated GFR.


Figure 9
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  		Fig. 9. Comparison of urinary megsin level according to the stage of diabetic nephropathy. Seventy-one diabetic patients positive for urinary megsin were classified into four stages by their proteinuria and estimated GFR (Materials and Methods). Proteinuria gradually increased in the patients at the advanced stage and the estimated GFR decreased after stage 3A, especially stage 3B. Of note, urinary megsin excretion increased in the patients as the stage of diabetic nephropathy progressed (up to stage 3A). When the disease advanced to stage 3B, urinary megsin levels decreased. Urinary megsin content did not correlate with proteinuria or the estimated GFR. The data are presented as mean ± SE. *P < 0.01 and **P < 0.001 compared with the stage 1 group; ***P < 0.05, +P < 0.01 and ++P < 0.001 compared with the stage 2 group; +++P < 0.05, {dagger}P < 0.01 and {ddagger}P < 0.001 compared with the stage 3A group; #P < 0.001 compared with the stage 3B group. eGFR; estimated GFR.

 


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
While our previous studies suggested usefulness of evaluation of megsin in biopsy samples, a non-invasive method to estimate expression of megsin is more desirable. ELISA of urinary megsin is ideal for this purpose. We therefore raised polyclonal and monoclonal antibodies to megsin and established a high-sensitive and megsin-specific sandwich ELISA. This ELISA system demonstrated that urinary megsin level is undetectable in most of the healthy subjects or patients with non-renal diseases. The existence of megsin in the urine was confirmed by the analysis of highly concentrated urine from urinary megsin-positive healthy individuals utilizing SDS–PAGE followed by TOF-mass spectrometry.

The molecular weight of megsin (~45 kDa) is below that of albumin, therefore, megsin in circulating blood is supposed to pass the glomerular filtration barrier and be excreted into urine. Plasma megsin levels in healthy volunteers or patients with renal disease were below the detection limit of the ELISA (data not shown). In contrast, some renal disease patients showed an increase in urinary megsin excretion. Thus, we speculate that urinary megsin may reflect local production of megsin in glomeruli and glomerular damage. Our speculation was supported by the results that urinary megsin level did not correlate with the disturbance of tubular absorption estimated by ß2-microglobulin excretion level.

Although the histological association with megsin excretion was not tested in this study, we demonstrated that urinary megsin excretion increased in accordance with stage progression of diabetic nephropathy, followed by the eventual decrease in the advanced stage. This evidence together with our previous studies [4,8,9] may also suggest an increase in megsin production in glomeruli. Further assessment of correlation between urinary megsin and proteinuria, glomerular function or pathological findings will reveal the diagnostic efficacy of megsin ELISA.

In conclusion, we established a sensitive megsin ELISA, which detects urinary megsin in some patients with renal diseases. Megsin ELISA may be a novel diagnostic tool for renal diseases.



   Acknowledgments
 
We thank Drs Daisuke Suzuki, Takatoshi Kakuta, Masanobu Miyazaki, Satoshi Sugiyama, Atsuhiro Yoshida, Hirokazu Imai and Ryuichi Furuya for collecting human subjects and Dr Takahisa Hachiya, Medical & Biological Laboratories Co., Ltd, and Ms Akiko Hirakawa for their technical support. This study is supported by a grant the New Energy and Industrial Technology Development Organization in Japan.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Schlondorff D. Roles of the mesangium in glomerular function. Kidney Int (1996) 49:1583–1585.[Web of Science][Medline]
  2. Miyata T, Nangaku M, Suzuki D, et al. A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy. J Clin Invest (1998) 102:828–836.[Web of Science][Medline]
  3. Inagi R, Nangaku M, Miyata T, et al. Mesangial cell-predominant functional gene, megsin. Clin Exp Nephrol (2003) 7:87–92.[CrossRef][Medline]
  4. Suzuki D, Miyata T, Nangaku M, et al. Expression of megsin mRNA, a novel mesangium-predominant gene, in the renal tissues of various glomerular diseases. J Am Soc Nephrol (1999) 10:2606–2613.[Abstract/Free Full Text]
  5. Inagi R, Miyata T, Suzuki D, et al. Specific tissue distribution of megsin, a novel serpin, in the glomerulus and its up-regulation in IgA nephropathy. Biochem Biophys Res Commun (2001) 286:1098–1106.[CrossRef][Web of Science][Medline]
  6. Nangaku M, Miyata T, Suzuki D, et al. Cloning of rodent megsin revealed its up-regulation in mesangioproliferative nephritis. Kidney Int (2001) 60:641–652.[CrossRef][Web of Science][Medline]
  7. Li YJ, Du Y, Li CX, et al. Family-based association study showing that immunoglobulin A nephropathy is associated with the polymorphisms 2093C and 2180T in the 3' untranslated region of the Megsin gene. J Am Soc Nephrol (2004) 15:1739–1743.[Abstract/Free Full Text]
  8. Miyata T, Inagi R, Nangaku M, et al. Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion. J Clin Invest (2002) 109:585–593.[CrossRef][Web of Science][Medline]
  9. Inagi R, Yamamoto Y, Nangaku M, et al. A severe diabetic nephropathy model with early development of nodule-like lesions induced by megsin overexpression in the RAGE/iNOS transgenic mice. Diabetes (2006) 55:356–366.[Abstract/Free Full Text]
  10. Ishikawa E, Imagawa M, Hashida S, et al. Ueno T. Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J Immunoassay (1983) 4:209–327.[Web of Science][Medline]
  11. Lamb EJ, Tomson CR, Roderick PJ. Clinical Sciences Reviews Committee of the Association for Clinical Biochemistry. Estimating kidney function in adults using formulae. Clin Chem (2006) 52:5–18.[Abstract/Free Full Text]
  12. Nochi T, Yuki Y, Terahara K et al. Biological role of Ep-CAM in the physical interaction between epithelial cells and lymphocytes in intestinal epithelium. Clin Immunol (2004) 113:326–339.[CrossRef][Web of Science][Medline]
Received for publication: 20.12.06
Accepted in revised form: 24. 5.07


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