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NDT Advance Access published online on October 16, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn576
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Characterization of the transcriptional regulation of the human MT1-MMP gene and association of risk reduction for focal-segmental glomerulosclerosis with two functional promoter SNPs

Astrid Munkert1, Udo Helmchen2, Markus J. Kemper3, Michael Bubenheim4, Rolf A. K. Stahl1 and Sigrid Harendza1

1 III. Medizinische Klinik 2 Nierenstiftung am 3 Klinik für Kinder- und Jugendmedizin 4 Institut für Medizinische Biometrie und Epidemiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany

Correspondence and offprint requests to: Sigrid Harendza, III. Medizinische Klinik, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. Tel: +49-40428033908; Fax: +49-40428035186; E-mail: harendza{at}uke.de



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest statement.
 References
 
Background. The matrix metalloproteinase MT1-MMP (MMP-14) is an important player in wound healing, bone development, angiogenesis, inflammation and tumour invasion. MT1-MMP also plays an important role in the development and resolution of experimental kidney diseases. The role of MT1-MMP was investigated for distinction between minimal-change glomerulonephritis (MCGN) and focal-segmental glomerulosclerosis (FSGS) that can sometimes be difficult due to sampling error in renal biopsy.

Methods. We defined the transcriptional regulation of the human MT1-MMP and the influence of single nucleotide polymorphisms (SNPs) within its promoter region in renal mesangial cells with reporter gene constructs and gel sift analysis. Genomic DNA from healthy blood donors (n = 500) and from kidney biopsies with defined renal diseases (MCGN: n = 189, FSGS: n = 311) was screened for MT1-MMP promoter SNPs.

Results. Transcription of MT1-MMP is regulated by two enhancers, an Sp1 binding site and a regulatory region 1 (RR1). RR1 contains an Ets site binding the transcription factors Elf-1 and E1AF but not NFAT. The MT1-MMP promoter contains two SNPs (–378 T/C and –364 G/T) in close vicinity to the RR1. Occurrence of the SNP variant –378 C leads to strong inhibition of nuclear protein binding to the RR1 reducing its enhancer function. Appearance of either variant –378 C or variant –364 T in at least one copy of the MT1-MMP promoter was associated with a significant risk reduction for the development of FSGS (P < 0.048).

Conclusion. Genetic testing for MT1-MMP promoter SNPs could put renal biopsy results into new perspective. An independent study will be required to verify these findings and their possible diagnostic value for differentiation between certain renal diseases.

Keywords: FSGS; glomerulonephritis; MT1-MMP; SNP; transcription



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Conflict of interest statement.
 References
 
The extracellular matrix (ECM) is a tightly regulated network of macromolecules. The main physiological regulators of ECM turnover are the matrix metalloproteinases (MMP) [1]. Synthesis, deposition and degradation of ECM occur during many physiological processes such as embryogenesis, angiogenesis and wound repair [2]. Excessive breakdown of the ECM is associated with rheumatoid arthritis and osteoarthritis, as well as tumour invasion and metastasis [3,4], whereas increased ECM deposition occurs in fibrotic diseases such as scleroderma and glomerulosclerosis [5,6]. Renal mesangial cell (MC) proliferation, increased matrix turnover and accumulation of interstitial collagens are common findings in most forms of progressive glomerular diseases, eventually leading to glomerular scarring in many cases [7]. Also, in experimental kidney diseases such as the anti-Thy-1.1 nephritis in rats, MC proliferation, accumulation of ECM proteins and increased expression of MMP occur as prominent features [8,9].

The membrane-type-1 MMP (MT1-MMP, MMP14) is one of six currently known MT-MMPs [10]. It is the predominant MT-MMP in the kidney and mainly produced by rat and human MC [11,12]. Besides degrading ECM components, MT1-MMP is the main activator of MMP-2 [13] that plays an important role in human and experimental kidney diseases [8,9,14]. In addition, MT1-MMP also represents a relevant component of the renal slit diaphragm [15]. Disruption of this delicate structure can cause the loss of podocyte foot processes and lead to nephrotic proteinuria [16] that occurs in minimal-change glomerulonephritis (MCGN) [17] and focal-segmental glomerulosclerosis (FSGS) [18].

Given the central role of MT1-MMP in these disease processes, little is known about its transcriptional regulation. Lohi et al. [19] found an enhancer function for the transcription factor Sp1 in MT1-MMP regulation in human fibrosarcoma cells. In Von Hippel–Lindau renal cell carcinoma, a HIF2-{alpha}-induced regulation of MT1-MMP was investigated with focus on two potential binding sites [20]. The transcription factors NFATc1, Sp1 and Sp3 as well as Egr-1 play an important role in the regulation of mouse MT1-MMP [21,22]. In this study, we lead an extensive investigation of the transcriptional regulation of the human MT1-MMP gene including an analysis of single nucleotide polymorphisms (SNPs) in its promoter region. In particular, we engaged in a systematic analysis whether SNPs in the MT1-MMP promoter affect its transcriptional regulation and are associated with renal diseases. Hence patients with MCGN and FSGS are screened for such SNPs with respect to alteration of relative risk for occurrence of these diseases.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Conflict of interest statement.
 References
 
Cell culture
Rat MCs were a kind gift of David Lovett, San Francisco VAMC/University of California. Cells were maintained in RPMI 1640 (GIBCO) supplemented with 10% fetal calf serum, 1% sodium pyruvate, 100 µg/ml streptomycin, 100 units/ml penicillin and 1.5% HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.8) buffer.

Human MT1-MMP promoter constructs
A set of 5'-deletion constructs of the human MT1-MMP promoter was prepared by PCR and directionally subcloned into the promoterless luciferase expression vector pGL3-Basic (Promega) using the KpnI-BglII sites. The resulting constructs were terminated at bp –1246, –808, –586, –344 and –202 relative to the translational start site and were denoted as pGL3-Luc 1246, pGL3-Luc 808, pGL3-Luc 586, pGL3-Luc 344 and pGL3-Luc 202, respectively. To test the functional significance of the –378 T/C and –364 G/T polymorphisms, site-specific mutagenesis was performed on wildtype genomic templates. These constructs were denominated pGL3-Luc 586 C378G364, pGL3-Luc 586 T378T364 and pGL3-Luc 586 C378T364. To test the interaction of Sp1 and RR1, binding sites were replaced by nonsense mutations and the constructs were denominated pGL3-Luc 586 RR1 neg, pGL3-Luc 586 RR1 neg Sp1 neg, pGL3-Luc 586 C378G364 Sp1 neg and pGL3-Luc T378G364586 Sp1 neg.

Transient transfection and luciferase activity
Transient transfections of rat MCs were performed with polyethyleneimine according to Boussif et al. [23]. The respective pGL3-Luc expression plasmids and a normalizing pCMV-β-galactosidase plasmid were used in concentrations of 2 µg/well. Total incubation time after transfection was 22–24 h. All experiments were carried out in triplicate and performed independently at least three times. Luciferase and β-galactosidase assays of cell lysates were performed as described [24,25]. Results are expressed as the ratio of luciferase activity to β-galactosidase activity.

Preparation of nuclear protein extracts
Nuclear extracts from rat MCs were prepared with a protocol adapted from Shaw et al. [26]. Cells were washed once with phosphate-buffered saline, harvested and resuspended in 1 ml buffer A [25 mM HEPES, 25 mM KCI, 5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), proteinase inhibitor Mix M (Serva, Heidelberg) and 1 mM ortho-vanadate]. Cells were lysed by adding half of cellular volume buffer A with 1.5% NP-40 (Sigma-Aldrich, Steinheim). The cell homogenate was centrifuged at 10 000 rpm for 30 s (HERAEUS 3325B rotor). The crude nuclear pellet was suspended in buffer C [50 mM HEPES [pH 7.6], 50 mM KCI, 0.1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10% glycerol], to a final volume of 315 µl. Thirty-five microlitres 3 M ammonium sulfate was added to a final concentration of 0.3 M for lysis of the nuclei. DNA was pelleted at 93 000 rpm for 10 min at 4°C (Eppendorf 5418D). The supernatant was recovered, and an equal volume of 3 M ammonium sulfate was added dropwise. Precipitated proteins were collected by centrifugation at 47 000 rpm for 10 min at 4°C (Eppendorf 5418D). The protein pellet was suspended in buffer C. The protein suspension extract was cleared by centrifugation through a YM-10 column (AMICON, Munich) at 13 000 rpm for 40 min at 4°C (HERAEUS 3325B rotor), washed with 70 µl buffer C per column, centrifuged for another 30 min and eluted from the column with 60 µl buffer C.

Electrophoretic mobility shift assay (EMSA)
Synthetic oligonucleotides were annealed and end labelled with polynucleotide kinase and [{gamma}-32P]dATP according to standard methodology. Ten micrograms of nuclear proteins were used in binding buffer (18 mM HEPES [pH 7.9], 200 nM EDTA, 100 nM EGTA, 40 nM NaCl, 200 nM DTT, 100 nM PMSF) with 2 µg of poly(dI-dC). They were incubated for 30 min at room temperature with 1 µl of the radiolabelled oligonucleotide (100 000 cpm/µl). Samples were electrophoresed on 4% polyacrylamide and 30% w/v glycerol gels in a buffer containing 1 x Tris borate/EDTA followed by autoradiography. For competition experiments, a 50- to 500-fold excess of unlabelled oligonucleotides was added to the reaction mixture as described above. Supershift experiments were performed by incubation of the ready reaction mixture overnight at 4°C with 4 µg/reaction of mouse monoclonal anti-human NFATc1 (7A6), goat polyclonal anti-human NFATc1 (K-18), rabbit polyclonal anti-human Elf-1 (C-20), rabbit polyclonal anti-human PEA3 (H-120) or control rabbit polyclonal anti-human p65 (C-20) IgG (Biotechnology, Santa Cruz) prior to addition of labelled oligonucleotide and electrophoresis.

Isolation of genomic DNA
Blood was collected from 500 healthy Caucasian blood donors and healthy children from Northern Germany. Genomic DNA was extracted using the Nucleo Spin Blood Quick Pure kit (Macherey and Nagel, Düren). Kidney biopsy samples from 189 MCGN and 311 FSGS patients from Caucasian origin were provided as a kind gift of Udo Helmchen. Genomic DNA from kidney biopsies was gained from a 30 µm slice of each paraffin-embedded biopsy with the DNeasy Blood & Tissue kit (Macherey and Nagel, Düren). This study was approved by the ethics committee of the physicians’ board of the city of Hamburg, and appropriate informed consent was obtained from human subjects involved.

SNP screening of the regulatory region 1 (RR1)
All 189 patients with MCGN (0–59 years of age) as well as 311 patients with FSGS (0–71 years of age) were matched with controls 1:1 stratifying for age and gender. To determine the diplotypes of patients and controls concerning the two SNPs next to the RR1 (–378 T/C and –364 G/T), a 495 bp fragment of the human MT1-MMP 5'-regulatory region spanning bp –585 to –90 was amplified by PCR using the BD Advantage 2 PCR System (Takara BIO, Potsdam) and the flanking primer pair 5'-GCCACATAGCCCCCAATAAT-3' and 5'- AGATCTTTGTCTTCGGTA-3'. PCR was carried out with 1 µl of genomic DNA from controls and biopsies, respectively, with the following protocol: 2 min at 95°C followed by 35 cycles (30s at 95°C, 30s at 61°C, 40s at 68°C) and a final 1 min at 68°C. PCR products were purified and sequenced with a nested primer 5'-GAACTGGGGTCTGGTT-3' in high throughput 96-well plates using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City) on an ABI3730 automated DNA sequencer. Allelic frequencies and Hardy–Weinberg distributions were calculated using standard methodologies.

Statistical analysis
For luciferase assay results, statistical significances were determined for paired comparisons using Student's t-test or by analysis of variance for multiple comparisons where appropriate. For the SNP screening, the {chi}2-test for homogeneity was used as a global test, allowing a margin of error of 0.05. Odds ratios were calculated in comparison to the reference group of homozygous wildtypes. Ninety-five percent confidence intervals (CI) were calculated using standard methodologies. Odds ratios with a 95% CI excluding one were regarded as statistically significant. Statistical levels of significance for deviation from expected Hardy–Weinberg distributions were determined by {chi}2-analysis.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Conflict of interest statement.
 References
 
Characterization of the transcriptional regulation of the human MT1-MMP gene and identification of SNPs
Transcription experiments revealed constitutive basal promoter activity up to bp –288 5' of the translational start site. Construct pGL3-Luc 586 expressed a 12-fold increase in luciferase activity in comparison with construct pGL3-Luc 288 (Figure 1). This region between bp –288 and bp –586 includes an Sp1 binding site [19] and an RR1. The Sp1 site between bp –101 and –92 [19] is highly conserved to the mouse sequence (bp –285 to –276 [21]), and the RR1 from bp –375 to –369 is highly conserved to bp –358 to –352 in the mouse MT1-MMP promoter [21]. A database search (Ensemble, NIH dbSNP and TESS) with 1.3 kb of the MT1-MMP promoter (NT_026437 [GenBank] , region: 4305633–4316643) and a correlation of SNPs with potential transcription factor binding sites resulted in 9 SNP entries and 315 calculated transcription factor binding sites. Two potentially important SNPs flanking RR1 were discovered: –364 G/T (rs1003349: G>T) and –378 T/C (rs1004030: T > C), revealing four possible haplotypes.


Figure 1
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  		Fig. 1 Transcriptional analysis of 5' deletion constructs of the human MT1-MMP promoter. Nucleotide positions with respect to the translational start site are given in the name of the constructs. Bars are the ratios of luciferase versus β-galactosidase activities, arbitrarily set equal to 1 for pGL3-Luc 288. Values are mean (± SEM) of at least nine independent experiments. *Significant increase in luciferase activity (P < 0.05).

 
Specificity of transcription factor binding
Using EMSA, we examined whether the SNP variants had any influence on the binding of nuclear proteins to the RR1 (Figure 2). With nuclear extracts from MC, the wildtype (Wt, T378G364) displayed one major retarded band (long arrow, lane 2) and a less prominent, second band (short arrow, lane 2) compared to the free oligonucleotide without protein (lane 1). Haplotype M1 (C378G364) displayed potent reduction of protein binding resulting in an almost complete extinction of the upper band (short arrow, lane 4) and considerable attenuation of the lower band (long arrow, lane 4). Haplotype M2 (T378T364) exhibited almost no signal alteration (lane 6) compared to the wildtype. Haplotype M3 (C378T364) led to slight diminution of signal intensity in the upper band (short arrow, lane 8) while the lower band remained nearly unaffected. Specificity of the discovered DNA-protein complex formation was tested in EMSA competition experiments. Figure 3 reveals that a 50-fold excess of the respective unlabelled oligonucleotide resulted in complete inhibition of complex formation with the radioactively labelled wildtype oligonucleotide (lane 3). A non-specific probe did not compete for binding proteins at this site (lane 6).


Figure 2
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  		Fig. 2 Haplotype-specific binding of nuclear proteins. EMSA analysis of allele-specific effects of the four different haplotypes Wt and M1–M3 on the interaction with nuclear proteins extracted from MC. Radiolabelled oligonucleotides (200 fmol) were incubated with 10 µg of nuclear extract from MC. The long arrow indicates a major shifted protein/DNA complex, and the short arrow indicates a minor shifted protein/DNA complex.

 

Figure 3
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  		Fig. 3 Specificity of nuclear protein binding. EMSA analysis with the wildtype (Wt 200 fmol) and nuclear proteins from MC (10 µg) was performed. The shifted double band (marked by the two arrows) is visible as in Figure 2, and competition experiments were carried out with 50- to 500-fold molar excess of unlabelled specific probe (lanes 3–5) and 50-fold molar excess of unlabelled non-specific probe (lane 6).

 
Influence of SNPs on transcriptional activity of RR1 and deletion constructs
Furthermore, the functionality of the SNPs with regard to Sp1- and RR1-regulated transcriptional activity of MT1-MMP was tested (Figure 4). With construct pGL3-Luc 344, a 5.5-fold increase in luciferase activity could be accredited solely to the segment comprising the Sp1 binding site compared to basal promoter activity. The RR1 wildtype (pGL3-Luc 586 T378G364) more than doubled the activity of pGL3-Luc 344. Construct pGL3-Luc 586 C378G364 caused a significant inhibition of this signal while pGL3-Luc 586 T378T364 and pGL3-Luc 586 C378T364 did not differ significantly from the activity of the wildtype construct (pGL3-Luc 586 T378G364). Site-directed mutagenesis leads to deletion constructs with the loss of binding capability in the Sp1 and RR1 regions, independent of the SNP variants. Activity levels of these constructs are shown in Figure 5. Destruction of the Sp1 binding site (pGL3-Luc 586 T378G364 Sp1 neg) leads to an ~75% reduction of luciferase activity, loss of the RR1 site (pGL3-Luc 586 RR1 neg) and to a 50% decrease in wildtype construct activity (pGL3-Luc 586 T378G364). The construct with the SNP variant –378 C within the normal context of RR1 and Sp1 (pGL3-Luc 586 C378G364) showed a loss of luciferase activity similar to a complete destruction of the RR1 binding site (pGL3-Luc 586 RR1 neg). An additional deletion of the Sp1 site within this construct (pGL3-Luc 586 C378G364 Sp1 neg) leads to a further drop of luciferase activity to levels comparable with activity of the basal promoter which corresponds to the activity of the double deletion construct (pGL3-Luc 586 RR1 neg Sp1 neg), where RR1 and Sp1 are replaced by nonsense mutations.


Figure 4
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  		Fig. 4 Transcriptional analysis with reporter constructs of the human MT1-MMP promoter with the four different genetic variants with regard to the SNPs –364 G/T and –378 T/C. Nucleotide positions with respect to the translational start site and genetic variants are given in the name of the constructs. Bars are the ratios of luciferase versus β-galactosidase activities, arbitrarily set equal to 1 for pGL3-Luc 288. Values are mean (± SEM) of at least nine independent experiments. *Significant decrease in luciferase activity (P < 0.05).

 

Figure 5
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  		Fig. 5 Transcriptional analysis with reporter constructs of the human MT1-MMP promoter with complete deletions (marked by an X) of the Sp1 binding site and/or the RR1 respectively and the SNP variant –378 C. Nucleotide positions with respect to the translational start site and specific deletion of respective binding sites or genetic variant are given in the name of the constructs. Bars are the ratios of luciferase versus β-galactosidase activities, arbitrarily set equal to 1 for pGL3-Luc 586 C378G364 Sp1 neg. Values are mean (± SEM) of at least nine independent experiments.

 
Screening of patients with the MCGN and FSGS for SNPs –378 T/C and –364 G/T
Since RR1 revealed binding of several transcription factors and the SNPs –378 T/C and –364 G/T lead to significant reduction in transcriptional activity of MT1-MMP, we wanted to study the impact of these SNPs on renal diseases with a disturbed balance in ECM turnover. A screening for these SNPs was performed for patients with MCGN and FSGS in comparison with healthy blood donors and healthy children. Allele frequencies of the SNPs –378 T/C and –364 G/T were in Hardy–Weinberg equilibrium in all groups, and no differences in distribution of the different genotypes were seen between patients and healthy controls (Table 1).


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  		Table 1 Allele and genotype frequencies of SNPs –378 T/C and –364 G/T in controls, MCGN and FSGS patients

 
Diplotype frequencies, i.e. the various combinations of SNPs –378 T/C and –364 G/T haplotypes on both human chromosomes, showed no differences for patients with MCGN ({chi}2 = 8.56; P = 0.479) compared with controls. However, FSGS patients showed statistically significant differences in comparison with controls ({chi}2 = 17.06; P = 0.048). Calculated odds ratios (Table 2) revealed that the diplotypes Wt/M1 (T378G364/C378G364), Wt/M2 (T378G364/T378T364), M1/M1 (C378G364/C378G364), M1/M2 (C378G364/T378T364) and M2/M2 (T378T364/T378T364) correlated with a significantly reduced risk of the occurrence of FSGS in comparison with the reference group of homozygous wildtypes (Wt/Wt).


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  		Table 2 Relative risk for disease displayed as odds ratios of diplotype constellations in MCGN and FSGS patients

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Conflict of interest statement.
 References
 
Increased MT1-MMP expression, leading to MMP-2 activation, is an important feature of glomerular inflammatory disease processes and is directly connected with the development of sclerosis and the loss of renal function [9,27]. Our study of the human MT1-MMP promoter region revealed besides basal constitutive promoter activity two transcriptionally active sites, an Sp1 site and a RR1. The Sp1 site is highly conserved in the mouse MT1-MMP gene where it displays functional relevance for transcriptional regulation of this gene irrespective of species or cell type [19,21]. In contrast to these findings, RR1 seems to play a species and cell-type-specific regulatory role for MT1-MMP even though its sequence is highly conserved. Its core sequence (bp –357 to –353) revealed specific binding and activation by NFATc for the mouse MT1-MMP gene [31]. Our supershift experiments with the RR1 revealed no supershifted band for NFATc1 but specific binding to the transcription factor proteins Elf-1 and E1AF that bind to the Ets consensus sequence EBS (A/TTCC) resembling the core of RR1 (data not shown) [28]. Since other factors of the Ets family such as Ets-1 and PU.1 play a role for MMP-2 regulation [29,30], further functional analysis will be needed to investigate the species-specific regulation of MT1-MMP.

Similar differential binding affinities have been described for Egr-1, Sp1 and Sp3 to an Sp1 site in the mouse MT1-MMP gene [21].

One (–378 T/C) of the two SNPs flanking, the RR1 was highly relevant for the transcriptional regulation of MT1-MMP. While the wildtype (–378 T) displayed a powerful enhancer function of RR1, the genetic variant (–378 C) leads to considerable loss in transcriptional activation (75%). This finding is of major significance since functional promoter SNPs are very rare in general [31], and two functional promoter SNPs adjacent to a half-palindromic estrogen receptor binding site and an Sp1 site within the human MMP-2 promoter have been found to significantly reduce MMP-2 activation [32]. Even though the variant –364 T revealed no significant reduction in transcriptional activation of MT1-MMP, it was not excluded from our further investigations.

Regarding the significance of the finely tuned transcriptional regulation of MT1-MMP and the functional relevance of the two SNPs flanking the RR1, the occurrence of the two SNPs was studied in patients with MCGN and FSGS. While no difference in diplotype distribution was found between patients with MCGN and controls, a significant reduction in the relative risk for occurrence of FSGS could be discovered for patients carrying either of the SNP variant –378 C or –364 T (M1 or M2) in at least one copy of their MT1-MMP gene.

In our in vitro data, occurrence of the homozygous wildtype is associated with a pronounced enhancer activity on the MT1-MMP promoter. An upregulation of MMP-2 activity in MCs is associated with an increased activity of MT1-MMP that leads to so-called epithelial–mesenchymal transformation (EMT) of renal cells, resulting in a proliferative, inflammatory phenotype and enhancement of the deposition of ECM at the same time [27,33,34]. This might be one mechanism maintaining the underlying persistent inflammatory process leading to a change of nomenclature from FSGS to FSSGN (focal-segmental sclerosing glomerulonephritis) underscoring the progression of this disease [35,36]. In many MCGN patients not responding appropriately to steroid treatment, a second kidney biopsy is performed often resulting in the diagnosis of FSGS in accordance with presumed sampling error in the first biopsy [37]. The risk reduction identified for patients suffering from FSGS and carrying either the SNP variant –378 C or –364 T is highly significant and might serve as an additional diagnostic tool for FSGS especially for patients with a high risk of complications in a second biopsy. No other genetic or humoral marker has been demonstrated to correlate with a significant risk reduction of occurrence of FSGS to date.

In summary, transcriptional regulation of the human MT1-MMP gene requires Elf-1 and E1AF as relevant trans-regulatory elements binding to the RR1 that is flanked by a functional SNP at bp –378 significantly affecting its transcriptional upregulation. The occurrence of SNP variants –378 C or –364 T within the genomic context furthermore leads to a significant reduction of the relative risk for FSGS. Further independent studies will be required to verify these findings and their possible diagnostic value in the differentiation of kidney diseases or their prognosis.



   Conflict of interest statement.
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest statement.
References
 
We declare that the results presented in this paper have not been published previously in whole or part, except in abstract format.



   Acknowledgments
 
We are grateful to Peter Kühnl, UKE, for providing blood samples from blood donors, and to Bettina Steinbach for excellent technical support. We thank David H. Lovett, San Francisco, for critical discussions. This work was supported by the Werner-Otto-Stiftung with a grant to Sigrid Harendza and a PhD scholarship for Astrid Munkert.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest statement.
 References
 

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Received for publication: 2. 7.08
Accepted in revised form: 22. 9.08


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