Alterations in the Renal Elastin-Elastase System in...

Alterations in the Renal Elastin-Elastase System in Type 1Diabetic Nephropathy Identified by Proteomic Analysis

VISITH THONGBOONKERD,*1 MICHELLE T. BARATI,* KENNETH R. MCLEISH,*†�

CHARAF BENARAFA,¶ EILEEN REMOLD-O’DONNELL,¶ SHIRONG ZHENG,‡

BRAD H. ROVIN,# WILLIAM M. PIERCE,§ PAUL N. EPSTEIN,‡§ andJON B. KLEIN*†�

*Core Proteomics Laboratory, Kidney Disease Program, Department of Medicine, and Departments of†Biochemistry and Molecular Biology, ‡Pediatrics, and §Pharmacology and Toxicology, University ofLouisville, Louisville, Kentucky; �Veterans Affairs Medical Center, Louisville, Kentucky; ¶Center for BloodResearch and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts; and #Department ofMedicine, Ohio State University School of Medicine, Columbus, Ohio

Abstract. Diabetes now accounts for �40% of patients withESRD. Despite significant progress in understanding diabeticnephropathy, the cellular mechanisms that lead to diabetes-induced renal damage are incompletely defined. For definingchanges in protein expression that accompany diabetic ne-phropathy, the renal proteome of 120-d-old OVE26 transgenicmice with hypoinsulinemia, hyperglycemia, hyperlipidemia,and proteinuria were compared with those of background FVBnondiabetic mice (n � 5). Proteins derived from whole-kidneylysate were separated by two-dimensional PAGE and identifiedby matrix-assisted laser desorption ionization–time-of-flight(MALDI-TOF) mass spectrometry. Forty-one proteins from300 visualized protein spots were differentially expressed indiabetic kidneys. Among these altered proteins, expression ofmonocyte/neutrophil elastase inhibitor was increased, whereas

elastase IIIB was decreased, leading to the hypothesis thatelastin expression would be increased in diabetic kidneys.Renal immunohistochemistry for elastin of 325-d-old FVB andOVE26 mice demonstrated marked accumulation of elastin inthe macula densa, collecting ducts, and pelvicalyceal epitheliaof diabetic kidneys. Elastin immunohistochemistry of humanrenal biopsies from patients with type 1 diabetes (n � 3)showed increased elastin expression in renal tubular cells andthe interstitium but not glomeruli. These results suggest thatcoordinated changes in elastase inhibitor and elastase expres-sion result in increased tubulointerstitial deposition of elastin indiabetic nephropathy. The identification of these coordinatedchanges in protein expression in diabetic nephropathy indicatesthe potential value of proteomic analysis in definingpathophysiology.

Diabetes now accounts for �40% of patients with ESRD, andthe number of renal failure patients with diabetes is expected toincrease in the coming years (1). Renal pathologic changes thatlead to decreased renal function are observed in all intrarenalstructures, including glomeruli, tubulointerstitium, and bloodvessels (2, 3). These morphologic changes, coupled with ele-vated intraglomerular pressure and hormonal dysregulation,lead to glomerulosclerosis, interstitial fibrosis, and ultimatelyrenal failure (4, 5). Despite recent progress in understanding

diabetic nephropathy, the cellular mechanisms that lead todiabetes-induced renal damage are incompletely defined.

Recently, Clarkson et al. (6) demonstrated that at least 200genes were differentially expressed in mesangial cells afterexposure to high-glucose media. These findings indicate thecomplexity of the development of diabetic nephropathy. How-ever, proteins, not genes, govern cellular functions. The studyof changes in renal protein expression is necessary to under-stand better the complex pathogenic mechanisms of diabetic ne-phropathy. Conventional protein studies—Western blotting andother immunologic methods—are limited to a relatively smallnumber of proteins that can be studied in each experiment and topreviously identified proteins for which specific antibodies areavailable. Proteomic analysis is an innovative approach that over-comes the limitations of immunology-based protein analyses. Weused proteomic analysis in the present study to evaluate globalchanges of renal protein expression in diabetic kidneys. Thediabetic animal model used in this study was the OVE26 trans-genic mouse model. OVE26 mice at 120 d of age displayed manycharacteristics of early-onset type 1 diabetic nephropathy, includ-ing hyperglycemia, hypoinsulinemia, hyperlipidemia, mesangialexpansion, and thickening of glomerular basement membrane(GBM) (7, 8). Because a protein database for mouse kidney was

Received August 1, 2003. Accepted December 6, 2003.1 Current address: Proteomics Center, Medical Molecular Biology Unit, Officefor Research and Development, Faculty of Medicine at Siriraj Hospital, Ma-hidol University, Bangkok, Thailand.Correspondence to Dr. Visith Thongboonkerd, Proteomics Center, MedicalMolecular Biology Unit, Office for Research and Development, 12th Floor—Adulyadej Vikrom Building, Siriraj Hospital, Prannok Road, Bangkoknoi,Bangkok 10700, Thailand. Phone: 66-2-4184793; Fax: 66-2-4184793; E-mail:[email protected]

1046-6673/1503-0650Journal of the American Society of NephrologyCopyright © 2004 by the American Society of Nephrology

DOI: 10.1097/01.ASN.0000115334.65095.9B

J Am Soc Nephrol 15: 650–662, 2004

not available, we created an initial renal proteome map for FVBnondiabetic mice (the background strain of the OVE26 line) andused this map as a reference to analyze protein expression indiabetic kidneys.

Comparison of protein expression in kidneys from OVE26and FVB mice by two-dimensional (2-D) PAGE demonstratedsignificant differences in expression levels of eight groups ofproteins: proteases, protease inhibitors, apoptosis-associatedproteins, regulators for oxidative tolerance, calcium-bindingproteins, transport regulators, cell signaling proteins, andsmooth muscle contractile elements. Our results showed coor-dinated changes in expression of monocyte/neutrophil elastaseinhibitor (MNEI), which was increased, and elastase IIIB,which was decreased. These findings suggested the hypothesisthat elastin, an extracellular matrix (ECM) protein, accumu-lates in diabetic kidneys and may participate in the develop-ment of diabetic nephropathy. This hypothesis was supportedby immunohistochemical studies in the OVE26 diabetic miceand patients with type 1 diabetes demonstrating increasedelastin deposition in renal tubular cells.

Materials and MethodsThe initial production, characterization, and maintenance of the

diabetic OVE26 line were performed at the University of Louisville asdescribed previously (7, 8). Control mice were nontransgenic animalsfrom the same strain (FVB). Ten animals (five in each group) werestudied. All animal studies were approved by the University of Lou-isville Institutional Animal Care and Use Committee and were inaccordance with NIH Guide for the Care and Use of LaboratoryAnimals.

Urine Albumin AssayMice (120 d old) were housed in metabolic cages (Nalgene, Brain-

tree, MA) with free access to solid laboratory food and feeding water.For obtaining adequate urine volume, the feeding water contained10% (vol/vol) Glucerna liquid diet (Abbotts Laboratories, Columbus,OH). Urinary albumin excretion on 24-h collections was measured bya commercial ELISA kit using goat anti-mouse albumin antibody(Bethyl Laboratories Inc., Montgomery, TX).

Extraction of Renal Proteins for 2-D PAGEMice were killed at 120 d of age by injection with Ketamine

HCl/Xylazine HCl solution (Sigma Chemical Co., St. Louis, MO).Protein extraction of the whole kidney was performed as describedpreviously (9, 10). Kidneys were frozen in liquid nitrogen; ground topowder; resuspended in a buffer containing 50 mM Tris, 0.3% SDS,and 200 mM DTT; and incubated at 100°C for 5 min. DNA and RNAwere removed by a buffer containing 500 mM Tris, 50 mM MgCl2, 1mg/ml DNAse I, and 0.25 mg/ml RNAse A. Excess salts wereremoved by acetone precipitation, and the protein pellet was finallyresuspended in a buffer containing 40 mM Tris, 7.92 M urea, 0.06%SDS, 1.76% ampholytes, 120 mM DTT, and 3.2% Triton X-100.Protein concentration levels were measured by spectrophotometryusing HP 8453 UV-visible system (Hewlett-Packard Company, PaloAlto, CA) and Bio-Rad Protein Assay (Bio-Rad Laboratories, Her-cules, CA).

First Dimension of 2-D PAGEA mobile ampholyte tube gel running system (Genomic Solutions

Inc., Ann Arbor, MI) was used for first-dimensional isoelectric fo-

cusing using 100 mM sodium hydroxide as the cathode buffer and 10mM phosphoric acid as the anode buffer. Precast carrier ampholytetube gels (pH 3 to 10), 1 mm � 18 cm, were prefocused with maximal1500 V and 110 �A per tube. Protein samples containing 100 �g fromindividual animals were loaded into individual tube gels and werefocused for 17 h and 30 min to reach 18,000 volt-hours.

Second Dimension of 2-D PAGEThe gels were extruded from the tubes after completion of focusing

and were incubated in premixed Tris/acetate equilibration buffer with0.01% bromophenol blue and 50 mM DTT for 2 min before loadingonto precast 10% homogeneous, 22 � 22-cm, slab gels (GenomicSolutions). The upper running buffer contained 0.2 M Tris base, 0.2 Mtricine, and 0.4% SDS, and the lower running buffer contained 0.625M Tris/acetate. Protein separation was performed with a maximum of500 V and 20,000 mW per gel.

SYPRO Ruby Staining and VisualizationThe gel slabs were fixed in 10% methanol and 7% acetic acid for

30 min. The fixed solution was removed, and 500 ml of SYPRO Rubygel stain (Bio-Rad Laboratories) was added to each gel and incubatedon gently continuous rocker at room temperature for 18 h. A high-resolution 12-bit camera with ultraviolet light box system (GenomicSolutions) was used to visualize the gel images.

Quantitative Analysis of Protein ExpressionInvestigator HT analyzer (Genomic Solutions) software was used

for matching and analysis of protein spots. The principles of mea-surement of intensity value by 2-D analysis software were similar tothose of densitometric measurement. Average mode of backgroundsubtraction was used to normalize intensity value that represents theamount of protein per spot. The normalized intensity values of indi-vidual protein spots were then used to determine differential proteinexpression between groups by statistical analyses.

Statistical AnalysesAfter completion of spot matching, spot intensities of each protein

spot from individual animals were compared between control anddiabetic groups. Because the sample size was relatively small, bothunpaired t test and Mann-Whitney U test by SPSS software v. 10.0were used for statistical analyses to avoid spurious results. P � 0.05was considered statistically significant. Only significant differencesthat were in agreement between the t test and the Mann-WhitneyU test were included, and the data were reported as mean � SEM.

In-Gel Tryptic Digestion, MALDI-TOF MassSpectrometry, and Peptide Mass Fingerprinting

In-gel tryptic digestion and matrix-assisted laser desorption ioniza-tion–time-of-flight (MALDI-TOF) mass spectrometry (MS) were per-formed using techniques described previously by our laboratory (11).Protein identification of peptide fragments was performed by us-ing the “ProFound” search engine (129.85.19.192/profound_bin/WebProFound.exe). The National Center for Biotechnology Informa-tion (NCBI) protein database was restricted to mammalian entries, andpeptides were assumed to be monoisotopic, oxidized at methionineresidues, and carbamidomethylated at cysteine residues. Up to onemissed trypsin cleavage was allowed, although most matches did notcontain any missed cleavages. A mass tolerance error of 150 ppm wasallowed for matching peptide mass values. Z scores were estimated bycomparison of search results against estimated random-match popu-

J Am Soc Nephrol 15: 650–662, 2004 Proteomics and Diabetic Nephropathy 651

lation and were the distances to the population mean in units of SD.Scores �1.65 were considered statistically significant (P � 0.05).Identities of protein spots that did not reach this significant level werenot reported.

Bioinformatic AnalysesTo examine potential protein function and sequence homology, we

performed bioinformatic analysis using public protein databases. In-ferred protein functions were determined by using data in the NCBI(www.ncbi.nlm.nih.gov/), Swiss-Prot, and TrEMBL protein databases(ca.expasy.org/sprot/). The similarity of amino acid sequences wasdetermined using the protein BLAST format (BLink) of the NCBIprotein database, as well as standard pairwise protein BLASTpsearches.

Western BlottingRenal proteins were mixed with 2� Laemmli sample buffer and

boiled for 5 min, and 30 �g of total proteins were loaded on 10%SDS-PAGE for MNEI Western blot. Proteins were transferred to anitrocellulose membrane and blocked with 5% milk/TTBS. The mem-brane was treated with rabbit polyclonal anti-MNEI serum (12) (1:1000 in 0.1% milk/PBS-Tween) at room temperature for 90 min. Thisserum cross-reacts with recombinant mouse elastase inhibitor A(EIA), the ortholog of MNEI (data not shown). Immunoreactiveprotein was detected by autoradiography using horseradish peroxida-se–conjugated antibody and chemiluminescent substrate.

ImmunohistochemistryImmunohistochemistry was performed on kidneys from 120- and

325-d-old FVB and OVE26 mice (n � 2) and on human renal biopsiesfrom patients with type 1 diabetes compared with normal biopsies,which were from candidate donors for renal transplantation (n � 3).Five-micrometer-thick mouse kidney sections were deparaffinizedand rehydrated. Antigen retrieval was performed by incubation inDAKO Target retrieval solution (DAKO Corp., Carpinteria, CA) at95°C for 20 min. Endogenous tissue peroxidase activity was sup-pressed by an incubation in 3% H2O2 at room temperature for 5 min.Nonspecific bindings were blocked using 5% (vol/vol) goat serum(Vector Laboratories, Burlingame, CA) in Tris-buffered saline atroom temperature for 1 h. The sections were then incubated withrabbit polyclonal anti-elastin antibody (#RDI-TP592; Research Diag-nostics Inc., Flanders, NJ), 1:400 in 1% goat serum at 4°C overnight.Slides were washed three times in Tris buffer before incubation withbiotinylated secondary antibody (Vector Laboratories), 1:200 in 1%goat serum at room temperature for 30 min. After three washes in Trisbuffer, the sections were incubated with an avidin/biotinylated per-oxidase complex (Vectastain Elite ABC kit; Vector Laboratories) for30 min. Immunoreactive elastin was detected by color developingwith Chromagen 3-3' diaminobenzidine (Vector Laboratories) for 4min. All sections were counterstained with hematoxylin. A section ofmouse aorta was used as the positive control. A negative control wasperformed by incubation with 1% goat serum without primaryantibody.

ResultsClinical Characteristics of OVE26 Diabetic Mice

Type 1 diabetes in the OVE26 transgenic model occurs as aresult of apoptosis of pancreatic � cells caused by overexpres-sion of the calcium-binding protein calmodulin (8). Metabolicderangements in OVE26 mice included hyperglycemia, hypo-

insulinemia, and hypertriglyceridemia. Hyperglycemia inOVE26 mice occurred within 1 wk after birth, and randomplasma glucose levels were �600 mg/dl from 10 wk of age.Plasma insulin levels of adult OVE26 mice were approxi-mately 30% of normal values as a result of the � cell–specific,calmodulin transgene (8). The mice typically survived withoutinsulin therapy or any other treatment for at least 1 yr. Mildmesangial expansion and GBM thickening were observed in120-d-old OVE26 diabetic mice without azotemia. For furthercharacterizing the renal phenotype of the OVE26 line, 24-hurinary albumin excretion was measured. OVE26 diabeticmice at 120 d of age had significantly increased 24-h urinaryalbumin excretion compared with the FVB controls (743 �461 versus 29 � 17 �g/24 h; P � 0.05). When the mice werekilled at approximately 17 wk (120 d), OVE26 mice had beendiabetic for approximately 16 wk.

Proteome Map of Normal FVB Mouse KidneyAs a database of mouse kidney proteins was not available, a

proteome map for FVB mouse kidney was produced. Renalproteins from individual animals (n � 5) were separated by2-D PAGE. The protein spot pattern was reproducible fromeach animal. Up to 300 protein spots were visualized on each2-D gel by SYPRO Ruby staining. Of these visualized proteinspots, 150 spots were excised and subjected to MALDI-TOFMS. The remaining spots were not excised, as their expressionwas likely below the threshold of detectability by MALDI-TOF MS. A total of 92 proteins were identified in our initialmouse kidney proteome map (Figure 1A). Positions of all ofthese identified proteins on 2-D gels were in the expected rangeof their theoretical isoelectric points (pI) and molecularweights (Mw). All identified proteins in the proteome map aresummarized in Table 1. Figure 2A illustrates mass spectrarepresentative of a single protein spot (spot 70 in Figure 1)obtained by MALDI-TOF MS, and Figure 2B demonstratespeptide mass fingerprint analysis that matched those massspectra with the mouse serine protease inhibitor EIA with afingerprint z score of 2.43 (�99 percentile; P � 0.01).

Differential Renal Protein Expression in the OVE26Mice during Early Type 1 Diabetic Nephropathy

Figure 1B shows a representative 2-D gel of 120-d-oldOVE26 mouse kidney proteins. A total of 41 protein spotswere differentially expressed in OVE26 diabetic kidneys com-pared with the controls (n � 5). Of these differentially ex-pressed proteins, 30 proteins were identified in normal mousekidney proteome map. The altered proteins were classified intofunctional groups on the basis of their major cellular functions,including proteases, protease inhibitors, apoptosis-associatedproteins, regulators of oxidative tolerance, calcium-bindingproteins, transport regulators, cell signaling proteins, andsmooth muscle contractile elements (Table 2). Some proteinshad variability in their expression levels among individualanimals. Therefore, visible differences of a number of proteinspots in Figure 1 were not included as significant changes.

652 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 650–662, 2004

Figure 1. The proteome maps of normal kidney from FVB mice (A) and diabetic kidney from OVE26 mice (B). Each map was created froma representative two-dimensional (2-D) gel image among five individuals in each group. Renal proteins were separated by 2-D PAGE on thebasis of differential isoelectric points (x axis) and molecular weights (y axis). Protein spots were excised and identified by matrix-assisted laserdesorption ionization–time-of-flight (MALDI-TOF) mass spectrometry (MS), followed by peptide mass fingerprinting. A total of 92 forms of65 unique proteins were identified in the normal kidney (summarized in Table 1). Renal protein expression in OVE26 kidneys was comparedwith the controls (n � 5). Protein spot pattern in diabetic kidneys was comparable to the normal kidneys. 2-D analysis software was used tomatch corresponding protein spots among gels, and the intensity of each spot was compared by statistical analyses described in the “Materialsand Methods” section. Expression levels of 41 protein spots were significantly changed in diabetic kidneys (summarized in Table 2). Of thesealtered protein spots, 30 proteins were identified in the proteome map, whereas the other 11 proteins (spots 93 to 103) were not identified. Thespots labeled with yellow-highlighted numbers were upregulated, whereas the spots labeled with blue-highlighted numbers were downregu-lated. Number labeling corresponds to the spot number in Tables 1 and 2.


Table 1. The identified proteins in the proteome map for normal FVB mouse kidneya

No. Proteins NCBI I.D. Accession

1 3-Mercaptopyruvate sulfurtransferase (MST) gi�3122930 P975322 Acidic nuclear phosphoprotein 32 gi�730318 P396873 Alpha enolase gi�13637776 P171824 Alpha-1-antitrypsin gi�203063 AAA407885 Alpha-1-antitrypsin gi�203063 AAA407886 Alpha-1-antitrypsin gi�203063 AAA407887 Alpha-1-macroglobulin gi�202857 AAA407238 Antithrombin-III gi�416621 P322619 Antithrombin-III gi�416621 P32261

10 Antithrombin-III gi�416621 P3226111 Antithrombin-III gi�416621 P3226112 Antithrombin-III gi�416621 P3226113 Apolipoprotein A-IV precursor gi�109575 B4089214 Apoliprotein A-I gi�109571 JC123715 Apoliprotein A-I gi�109571 JC123716 ATP synthase beta subunit gi�1374715 AAB0228817 ATP synthase beta subunit gi�1374715 AAB0228818 ATP synthase delta chain, mitochondrial precursor gi�1352036 P3543419 Calbindin gi�115396 P0717120 Calbindin gi�115396 P0717121 Calmodulin gi�223872 1003191A22 Calreticulin gi�117505 P1841823 Cellular retinol-binding protein gi�809309 80930924 Chain D, deoxyribonuclease I complex with actin gi�229691 22969125 Chloride intracellular channel protein 1 gi�6685328 Q9Z1Q526 Clathrin, light polypeptide (Lca) gi�7949023 NP_05804027 Collagen alpha 3 (V) chain gi�105709 S2037528 Complement component 1, q subcomponent binding protein gi�9506435 NP_06213229 Contraception associated protein 1 (CAP 1) gi�7429594 JE 034430 Crocalbin-like protein gi�8515718 AAF7614131 Cu/Zn superoxide dismutase gi�226471 22647132 Cu/Zn superoxide dismutase gi�226471 22647133 Cu/Zn superoxide dismutase gi�226471 22647134 Cytochrome b5 gi�554539 AAA7242035 Cytoskeletal tropomyosin gi�37424 CAA2825836 Cytoskeletal tropomyosin gi�37424 CAA2825837 Deoxyribonuclease I (Dnase I) gi�494869 49486938 Deoxyribonuclease I (Dnase I) gi�494869 49486939 Dna-K type molecular chaperone grp75 precursor gi�2119726 I5658140 Dna-K type molecular chaperone grp75 precursor gi�2119726 I5658141 Elastase IIIB (protease E) gi�14195655 P0886142 Ferritin heavy chain gi�6753912 NP_03436943 Ferritin heavy chain gi�6753912 NP_03436944 Ferritin heavy chain gi�6753912 NP_03436945 Ferritin light chain 1 gi�6753914 NP_03437046 Fructose 1,6-bisphosphatase gi�119740 P1911247 Glutamate cysteine ligase gi�8393446 NP_05900148 GRP78 (78 kDa glucose-regulated protein) gi�4033392 Q9059349 GRP78 (78 kDa glucose-regulated protein) gi�4033392 Q9059350 GTP-specific succinyl-CoA synthetase beta subunit gi�3766203 AAC6439951 Heat shock 60 kDa protein (HSP60) (60 kDa chaperone) gi�1334284 CAA3765452 Heat shock 60 kDa protein (HSP60) (60 kDa chaperone) gi�1334284 CAA3765453 High mobility group 1 protein gi�600761 AAA57042


Coordinated Changes in the Renal Elastin-ElastaseSystem in Diabetic Kidneys

We identified increased expression of the serine proteaseinhibitor EIA (spot 70, Figures 1 and 2). EIA was recentlyidentified as one of the four mouse homologs of the humanelastase inhibitor MNEI (13). MNEI is one of the most effi-cient inhibitors of elastase-like serine proteases and is theproduct of a single gene (SERPINB1) in humans (14, 15). Thecharacterization of the mouse homologs revealed that EIA is

the only functional counterpart of MNEI on the basis of se-quence, tissue expression, and inhibitory function (13). West-ern blot analysis in 120-d-old mouse kidneys confirmed thepresence of MNEI in normal kidneys and its upregulation bydiabetes (Figure 3). Therefore, MNEI and EIA are used inter-changeably in the rest of this article.

Coordinated changes in expression of elastase inhibitorMNEI, which was increased (Figure 4, A and C), and elastaseIIIB, which was decreased (Figure 4, B and D), suggested the

Table 1. (continued)

No. Proteins NCBI I.D. Accession

54 Hippocampal cholinergic neurostimulating peptide precursor gi�9453889 BAB0327655 Histone H3.2 gi�70755 HSXL3256 HSPC207 gi�7106804 AAF3612757 Ig VL gi�227745 22774558 Lactate dehydrogenase 2, B chain gi�6678674 NP_03251859 Myosin, light chain, smooth muscle gi�12737351 XP_01218060 NADH-ubiquinone oxidoreductase 24 kDa subunit precursor gi�128867 P1923461 NADH-ubiquinone oxidoreductase 75 kDa subunit precursor gi�128825 P1569062 Na-H exchanger, isoform 3 regulator 1 gi�6755566 NP_03616063 Na-H exchanger, isoform 3 regulator 1 gi�6755566 NP_03616064 Na-H exchanger, isoform 3 regulator 1 gi�6755566 NP_03616065 Na-H exchanger, isoform 3 regulator 1 gi�6755566 NP_03616066 Nucleobindin gi�16758210 NP_16758267 Phosphatidylethanolamine binding protein gi�8393910 NP_05893268 Put. Beta actin gi�49868 CAA2739669 Put. Beta actin gi�49868 CAA2739670 Serine protease inhibitor EIA; serpin clade B gi�22347578 AAM9593371 Recombinant rat annexin V gi�4139939 1BC172 Alpha 1-antitrypsin/serine protease inhibitor 1-1 gi�6678079 NP_03326973 Alpha 1-antitrypsin/serine protease inhibitor 1-1 gi�6678079 NP_03326974 Alpha 1-antitrypsin/serine protease inhibitor 1-1 gi�6678079 NP_03326975 Alpha 1-antitrypsin/serine protease inhibitor 1-1 gi�6678079 NP_03326976 Albumin gi�5915682 P0772477 Albumin gi�5915682 P0772478 Albumin gi�5915682 P0772479 Albumin gi�5915682 P0772480 Skeletal muscle tropomyosin gi�339958 AAA6122781 Sodium-hydrogen exchanger regulatory factor gi�5732682 AAD4922482 Syntaxin 11 gi�7447077 JE009483 Thioredoxin gi�135776 P1123284 T-kininogen gi�207341 AAA422585 Transthyretin gi�7305599 NP_03872586 Tropomyosin 1, smooth muscle gi�136100 P1046987 Tropomyosin 4 gi�6981672 NP_03681088 Tropomyosin 5 gi�136097 P2110789 Tropomyosin alpha chain, smooth muscle gi�136101 P0646990 Tropomyosin, fibroblast isoform 1 gi�1174753 P4690191 Tubulin, beta-3 gi�12852060 BAB2925792 Vimentin gi�1353212 P48670

a The identified proteins were summarized with the NCBI identification and accession numbers. Only the identities with significant zscores (�1.65, P � 0.05) were included. All of the identified proteins were in the expected ranges of pI and Mw in the 2-D gel in Figure1A.


Table 2. Quantitative analysis and functional classification of differentially expressed proteins in OVE26 diabetic mouse kidneya

Proteins Spot No.Normal DM Alteration

(DM/Normal)Average SEM Average SEM

Protease inhibitorsantithrombin-IIIb 12 21061 1982 36561 2971 Up (1.74)hippocampal cholinergic neurostimulating peptide precursorc,d 54 184971 27935 285705 23368 Up (1.54)serine protease inhibitor EIA (Serpinb1)e 70 27525 7505 77069 16540 Up (2.80)T-kininogenb 84 14506 2930 45489 12234 Up (3.14)

Proteasescontraception associated protein 1 (CAP 1)f,g 29 6658 5300 32339 21017 Up (4.86)elastase IIIB (protease E) 41 3377 884 1889 785 Down (0.56)

Apoptosis-associated proteinsdeoxyribonuclease I (Dnase I) 37 27650 4364 102206 18588 Up (3.70)histone H3.2 55 7848 3181 21115 7500 Up (2.69)

Regulators of oxidative toleranceferritin heavy chainb 42 6890 2164 27206 4661 Up (3.95)ferritin heavy chainb 44 89632 30177 172614 16286 Up (1.93)ferritin light chain 1b 45 373232 31457 561829 28061 Up (1.51)

Calcium-binding proteinscalbindinb 20 10538 9629 70654 53779 Up (6.70)calmodulinb 21 125072 43571 398045 139427 Up (3.18)crocalbin-like proteinh 30 2430 902 9598 4151 Up (3.95)recombinant rat annexin V 71 2978 465 7206 899 Up (2.42)

Transport regulatorscellular retinol-binding proteinb 23 33017 7247 69722 12705 Up (2.11)Na-H exchanger, isoform 3 regulator 1b 62 8095 1847 12810 937 Up (1.58)syntaxin 11 82 6142 2145 20802 7270 Up (3.39)transthyretinb 85 68362 4563 37306 7219 Down (0.55)

Cell-signaling proteinscomplement component 1, q subcomponent binding proteini 28 39722 11846 108044 36701 Up (2.72)

Smooth muscle contractile elementscytoskeletal tropomyosinb 35 383707 44003 730919 138816 Up (1.90)myosin, light chain, smooth muscleb 59 51242 12998 116637 39385 Up (2.28)skeletal muscle tropomyosinb 80 8244 1491 16826 1990 Up (2.04)tropomyosin 1, smooth muscleb 86 6614 1149 18950 4519 Up (2.86)tropomyosin 5b 88 4256 1011 12602 3543 Up (2.96)tropomyosin alpha chain, smooth muscleb 89 77722 18199 123246 16660 Up (1.59)tropomyosin, fibroblast isoform 1b 90 113846 29107 226989 79713 Up (1.99)vimentinb 92 8827 2112 24373 6454 Up (2.76)

Miscellaneousapolipoprotein A-IV precursorb 13 14698 3514 39656 13772 Up (2.70)HSPC207j 56 27175 4031 38678 7869 Up (1.42)unidentified 93 1326 569 2129 457 Up (1.61)unidentified 94 4267 818 6969 1319 Up (1.63)unidentified 95 13487 3108 21497 5025 Up (1.59)unidentified 96 3020 394 4447 509 Up (1.47)unidentified 97 3893 1106 16322 5095 Up (4.19)unidentified 98 2179 834 4517 1837 Up (2.07)unidentified 99 3911 539 7872 1332 Up (2.01)unidentified 100 1924 983 14320 8936 Up (7.44)unidentified 101 17652 2633 8395 1417 Down (0.48)unidentified 102 2700 1091 11030 3410 Up (4.09)unidentified 103 4630 4192 15224 8128 Up (3.29)

a The spot intensity (in pixel units) representing the amount of protein per spot was analyzed using 2-D analysis software. Onlystatistically significant changes were included, and the altered proteins were classified into variable functional categories on the basis oftheir major functions in the protein databases.

b Proteins that were previously shown to be regulated during diabetes.c 99% amino acid identity with phosphatidylethanolamine binding protein (gi�8393910, NP_058932).d Synonym: Raf kinase inhibitor protein (RKIP).e 80% amino acid identity with human monocyte/neutrophil elastase inhibitor (MNEI) (gi�266344, P30740).f 95% amino acid identity with DJ-1 (gi�3256343, BAA29063).g Containing a protease domain (merops.sanger.ac.uk).h 99% amino acid identity with calumenin (gi�14718453, AAK72908).i 95% amino acid identity with P32-RACK (receptor of activated protein kinase C) (gi�18652991, AAL77246).j 97% amino acid identity with uncharacterized bone marrow protein.


hypothesis that elastin expression would be increased in dia-betic kidneys. To determine whether elastin accumulated indiabetic kidneys and to define the location of this change, we

performed immunohistochemical study for elastin on mousekidneys and human renal biopsies. Kidneys from 120-d-old (n� 2) and 325-d-old (n � 2) mice were studied. Normaldistribution of elastin in mouse kidneys was observed in theBowman’s capsule, GBM, juxtaglomerular apparatuses, andvessels, with the most prominent staining in proximal tubularepithelial cells. Although there was no difference observed indiabetic mice versus normal at 120 d of age, a markedlyincreased accumulation of elastin was observed in the maculadensa, collecting ducts, and pelvicalyceal epithelia in diabetickidneys of older (325 d) mice (Figure 5).

Figure 6 shows the elastin immunohistochemistry of humanrenal biopsies (n � 3). Normal distribution of elastin in humankidneys was different from that in normal mice. In general,elastin expression in renal tubular cells and the glomeruli wasgreater in humans than in mice. Elastin expression was mark-edly increased in renal tubular epithelial cells in early-stagediabetic kidneys (Figure 6, B and D) and in the interstitium oflate-stage diabetic kidney (Figure 6F). There was no obviousincrease in the amount of elastin staining in the glomeruli ofpatients with diabetes, although the staining was more promi-nent in the periphery of glomerular tufts from patients withdiabetes.

DiscussionCurrent therapy aiming to halt the progression of renal

damage in established diabetic nephropathy is limited to anti-hypertensive drugs, especially angiotensin-converting enzymeinhibitors and angiotensin receptor blockers (16). Althoughthis therapy effectively slows the rate of progression of diabeticrenal injury, renal failure remains a common complication.Defining the pathophysiologic mechanisms of diabetic ne-phropathy is necessary to identify new targets for therapeuticintervention. Our approach to defining potential novel mecha-nisms of diabetic nephropathy was to identify proteins withaltered expression in diabetic kidneys using proteomic tech-niques. Developments in proteomic techniques during the pastdecade allow simultaneous identification of a large number ofproteins and comparison of expression of these proteins be-tween groups. The present study compared renal protein ex-pression of OVE26 mice, a transgenic mouse model that mim-ics many aspects of human type 1 diabetes, with that of FVBmice, a background nondiabetic strain. The renal lesion inOVE26 mice was characterized previously by showing renalhistologic changes commonly seen in human diabetic nephrop-athy, including mesangial expansion and increased thickness ofthe GBM by 120 d of age (7). The present study also showsthat OVE26 mice had a dramatic increase in urinary albuminexcretion. Thus, OVE26 mice present many of the featuresobserved in human diabetic nephropathy.

We identified 92 proteins in our initial proteome map ofnormal FVB mouse kidney, 30 of which were differentiallyexpressed in diabetic kidneys. The mechanism for alteredexpression of these proteins was not investigated but could beinduced by hyperglycemia and/or hypoinsulinemia, as the an-imals were both hyperglycemic and hypoinsulinemic. Nineteenof these altered proteins were previously shown to be regulated

Figure 2. MALDI-TOF MS and peptide mass fingerprinting. (A)Peptide masses (in mass per charge [m/z] units) were obtained byMALDI-TOF MS after in-gel tryptic digestion of spot 70 in Figure 1.(B) The peptide masses were queried to the theoretical masses ofmammalian entries in the National Center for Biotechnology Infor-mation protein database using the ProFound search engine. A maxi-mum 150-ppm error window and one missed tryptic cleavage wereallowed, although most of the matched masses had no missed cleav-age. After excluding autolytic trypsin masses, 10 of 12 sample massesmatched with serine protease inhibitor (EIA), serpin clade B with zscore of 2.43 (�99 percentile, P � 0.01). *Oxidation at methionineresidue.

Figure 3. Western blotting for monocyte/neutrophil elastase inhibitor(MNEI). Proteins derived from whole-kidney lysate of 120-d-old FVBand OVE26 mice (total 30 �g) were separated by 1-D PAGE andtransferred to a nitrocellulose membrane. Rabbit polyclonal anti-MNEI was used as a primary antibody. Only a single band at approx-imately 42 kD, the expected molecular size, was observed in eachlane. Recombinant human MNEI was used as a positive control.Western blot analysis confirmed that EIA, the murine ortholog ofMNEI, was present in the mouse kidney and was upregulated in theOVE26 diabetic kidneys.


during diabetes (4, 17–24) and are marked with a superscript bin Table 2. Involvement of the other 11 altered proteins indiabetic nephropathy had not previously been reported, sug-gesting their roles in novel mechanisms of diabeticnephropathy.

When we examined the list of proteins regulated in diabetickidneys (Table 2), the only apparent pathway in which multipleproteins were regulated was the elastin-elastase pathway. Be-cause the role of the elastin ECM protein had not been wellcharacterized in diabetic nephropathy, changes in elastin-reg-ulating proteins focused our attention on this pathway. Expres-sion of the elastase inhibitor MNEI was increased approxi-mately threefold in diabetic kidneys. MNEI is one of the mostefficient inhibitors of elastase-like serine proteases by formingstable covalent inhibitory complexes with target proteases.Whereas the expression of elastase inhibitor MNEI was in-creased in diabetic kidneys, elastase IIIB expression was de-creased. Thus, both expression and activity of elastase wouldbe predicted to be decreased in diabetic nephropathy. Thesecoordinated changes defined by proteomic analysis suggestedthe hypothesis that elastin expression would be increased indiabetic kidneys. Renal elastin immunohistochemistry was per-formed to address this hypothesis.

The elastin distribution in normal mouse kidney in thepresent study was similar to that observed by Sterzel et al. (25).Immunohistochemical analysis confirmed an increase in elastinexpression in diabetic kidneys, particularly in the macula

densa. The changes in elastin expression in 325-d-old mice butnot at 120 d suggested that a gradual increase in elastinexpression required long-term changes in MNEI and elastaseIIIB. The pattern of elastin expression in normal human kid-neys differed from that in normal mice with greater elastinexpression in both tubules and glomeruli in human kidneys. Inpatients with diabetes, elastin expression was increased in renaltubular epithelial cells. The mechanism of differences in nor-mal distribution and increased elastin expression in mice ver-sus humans remains unknown.

A role for elastin in the pathogenesis of diabetic nephropathyhas not previously been described. The major function ofelastin is to provide vascular wall elasticity (26). Alterations inelastin expression and function are associated with vasculopa-thy of large vessels induced by diabetes (27, 28). However, wedid not observe change in elastin deposition in the intrarenalvessels. Therefore, it is unlikely that altered elastin expressionobserved in our study is related to diabetic renal vasculopathy.In the kidney, elastin plays an important role in stabilizing theglomerular tuft (25). The increase in elastin expression, how-ever, was located in renal tubular epithelial cells, not glomer-uli. These findings suggest that elastin may play a role intubular or interstitial changes, which accompany diabeticnephropathy.

Elastin deposition is a highly regulated process that occursprimarily during early development (29). Increased elastinexpression results from increased transcription and translation.

Figure 4. Coordinated changes of elastase inhibitorMNEI (A and C) and elastase IIIB (B and D). (A and B)Zoom-in images of spots 70 and 41, respectively, fromindividual animals. (C and D) The summary of intensitydata of those two spots. The elastase inhibitor MNEIwas increased, whereas the elastase IIIB was decreasedin diabetic kidneys. *P � 0.05.


TGF-�, which is upregulated in diabetic kidneys, was reportedpreviously to stabilize elastin mRNA and promote elastin dep-osition (30). The increased elastin expression identified in

renal tubular epithelial cells, not in extracellular spaces, oc-curred in parallel with an increase of vimentin (Table 2), amarker for cells derived from mesenchymal tissues (31). Ourdata were consistent with the data reported by Rastaldi et al.(32) that renal tubular epithelial cells can produce ECM pro-teins and directly intervene in fibrotic processes via the epi-thelial-mesenchymal transdifferentiation. The increase in ex-pression of a myofibroblast protein (fibroblast tropomyosin)and proteins associated with proliferation, modulation, anddifferentiation of myofibroblast and fibrogenesis (calmodulinand cellular retinol-binding protein; Table 2) provides furthersupport for this process (33, 34). In addition, Figure 6F showsa marked increase of elastin staining in the tubulointerstitiumof an end-stage diabetic kidney. Taken together, these datasuggest that elastin may play a role in tubular disorders andinterstitial fibrosis in diabetic nephropathy. However, elastinaccumulation might reflect advanced tissue response to injury.Thus, alterations in the renal elastin-elastase system may be thecause of or the result of diabetic nephropathy. Further func-tional and time-course studies are required to evaluate thesignificance of disordered elastin deposition.

Although our study is the first to examine global changes inprotein expression in diabetic kidneys, genomic approacheswere previously applied to identify genes in renal cells regu-lated by hyperglycemia. Clarkson et al. (6) showed that 200genes were differentially expressed when mesangial cells werepropagated in high ambient glucose in vitro. It is not surprisingthat our proteomic data and Clarkson’s genomic data are notcompletely concordant. Some genes, such as ferritin, wereupregulated by hyperglycemia at the level of transcription andtranslation. However, other genes, such as myosin, were down-regulated in Clarkson’s study, but the protein products of thosegenes were upregulated in vivo in our OVE26 diabetic mice.Several differences in the experimental approaches might ac-count for the disparate findings. First, we studied the wholekidneys from diabetic animals, whereas Clarkson et al. studiedmesangial cells in vitro. Second, changes in mRNA and proteindo not always correlate (35–38). Third, differential proteinexpression can result from posttranslational modifications thatare not detectable by genomic analysis. Finally, protein deg-radation is not measured by changes in mRNA expression.

Several limitations of 2-D proteomic analysis in the presentstudy need to be noted. First, this approach identified high-abundance proteins, whereas detection of low-abundance pro-teins was limited. This may explain the failure to identifyseveral proteins previously shown to be regulated in diabetes,for example, TGF-� and protein kinase C. Second, we did notidentify all of the proteins present in the 2-D gels. Using moresensitive mass spectrometric techniques, such as tandem MS,may permit identification of low-abundance proteins. Third, alarger number of visualized protein spots would be expected onindividual gels than the 300 spots observed in the presentstudy. This limitation likely resulted from the small amount ofprotein (100 �g) used for individual analytical gels. In addi-tion, the extraction protocol used did not solubilize someprotein components, especially membrane-associated and hy-drophobic proteins.

Figure 5. Renal immunohistochemistry for elastin in 325-d-old mice.(A) Positive control from FVB mouse aorta demonstrates that elastinis typically present in intimal and adventitial layers of the aorta. (B)Negative control demonstrates the absence of elastin immunoreactivestaining. C, E, G, and I were from normal (FVB) mice, and D, F, H,and J were from diabetic (OVE26) mice. Normal distribution ofelastin was most prominent in proximal tubular epithelial cells (C). Inthe OVE26 diabetic kidneys, elastin expression was increased in themacula densa (D), collecting ducts (F), and pelvicalyceal epithelia(H). There was no change of elastin expression observed in the vessels(I and J). Magnification, �40.


We examined protein expression from the whole kidneys inthe present study. This approach cannot identify localizedchanges such as those that may be confined to glomeruli,tubules, or even podocytes. In additional, the magnitude ofchanges is affected by degrees of changes in individual intra-renal structures. For example, mild changes in mesangial cellsmay not be detected in the whole-kidney analysis if there is no

change in other structures or changes in other structures are inan opposite direction. Moreover, it should be noted that iden-tification of the altered proteins in the kidney does not confirmthat they are kidney-specific changes, as a systematic study ofother organs was not performed. Changes similar to those thatwe observed in the kidney may be present in liver, muscles, orother organs. Finally, we examined renal protein expression

Figure 6. Elastin immunohistochemistry of human renal biopsies. A, C, and E were from three normal biopsies, and B, D, and F were fromthree patients with type 1 diabetes. Elastin expression was markedly increased in renal tubular epithelial cells in early-stage (B and D) and inthe interstitium of late-stage (F) diabetic kidneys. There was no obvious increase in the amount of elastin staining in the glomeruli of patientswith diabetes, although the staining was more prominent in the periphery of glomerular tufts from patients with diabetes (B). Magnifications:�40 in A through E; �10 in F.


only in 120-d-old animals. Defining alterations in renal proteinexpression at a single time point does not represent the entiredynamic process of diabetic nephropathy. We are currentlyperforming a serial study of other time points of isolatedglomeruli and blood vessels to characterize more thoroughlythe renal subproteome during diabetes.

In summary, proteomic analysis revealed coordinatedchanges of elastase inhibitor MNEI and elastase IIIB, leadingto the identification of increased elastin expression in mouseand human diabetic kidneys. Application of proteomic analysismay yield new insights into the pathogenic mechanisms ofdiabetic nephropathy.

AcknowledgmentsThis study was supported by National Institutes of Health Grants

R21 DK62086-01 (J.B.K.), R01 HL66358-01 (J.B.K.), R21 DK06289(K.R.M.), and HL66548 (E.R.O.) and Department of Veterans Affairs(J.B.K. and K.R.M.). We gratefully acknowledge the assistance ofPatricia M. Kralik, Jian Cai, Xia Shen, Naira Meterveli, and MichaelE. Brier.

References1. USRDS. United States Renal Data System. Incidence and prev-

alence of ESRD. Am J Kidney Dis 30: S40–S53, 19972. Schleicher E, Kolm V, Ceol M, Nerlich A: Structural and func-

tional changes in diabetic glomerulopathy. Kidney Blood PressRes 19: 305–315, 1996

3. Mauer SM, Lane P, Hattori M, Fioretto P, Steffes MW: Renalstructure and function in insulin-dependent diabetes mellitus andtype I membranoproliferative glomerulonephritis in humans.J Am Soc Nephrol 2[Suppl 1]: S181–S184, 1992

4. Lehmann R, Schleicher ED: Molecular mechanism of diabeticnephropathy. Clin Chim Acta 297: 135–144, 2000

5. Marcantoni C, Ortalda V, Lupo A, Maschio G: Progression ofrenal failure in diabetic nephropathy. Nephrol Dial Transplant13[Suppl 8]: 16–19, 1998

6. Clarkson MR, Murphy M, Gupta S, Lambe T, Mackenzie HS,Godson C, Martin F, Brady HR: High glucose-altered geneexpression in mesangial cells. Actin-regulatory protein gene ex-pression is triggered by oxidative stress and cytoskeletal disas-sembly. J Biol Chem 277: 9707–9712, 2002

7. Carlson EC, Audette JL, Klevay LM, Nguyen H, Epstein PN:Ultrastructural and functional analyses of nephropathy in calm-odulin-induced diabetic transgenic mice. Anat Rec 247: 9–19,1997

8. Epstein PN, Overbeek PA, Means AR: Calmodulin-induced ear-ly-onset diabetes in transgenic mice. Cell 58: 1067–1073, 1989

9. Thongboonkerd V, Gozal E, Sachleben LR, Arthur JM, PierceWM, Cai J, Chao J, Bader M, Pesquero JB, Gozal D, Klein JB:Proteomic analysis reveals alterations in the renal kallikreinpathway during hypoxia-induced hypertension. J Biol Chem 277:34708–34716, 2002

10. Arthur JM, Thongboonkerd V, Scherzer JA, Cai J, Pierce WM,Klein JB: Differential expression of proteins in renal cortex andmedulla: A proteomic approach. Kidney Int 62: 1314–1321,2002

11. Thongboonkerd V, Luengpailin J, Cao J, Pierce WM, Cai J,Klein JB, Doyle RJ: Fluoride exposure attenuates expression ofStreptococcus pyogenes virulence factors. J Biol Chem 277:16599–16605, 2002

12. Rees DD, Rogers RA, Cooley J, Mandle RJ, Kenney DM,Remold-O’Donnell E: Recombinant human monocyte/neutrophilelastase inhibitor protects rat lungs against injury from cysticfibrosis airway secretions. Am J Respir Cell Mol Biol 20: 69–78,1999

13. Benarafa C, Cooley J, Zeng W, Bird PI, Remold-O’Donnell E:Characterization of four murine homologs of the human ov-serpin monocyte neutrophil elastase inhibitor MNEI (SER-PINB1). J Biol Chem 277: 42028–42033, 2002

14. Remold-O’Donnell E, Chin J, Alberts M: Sequence and molec-ular characterization of human monocyte/neutrophil elastase in-hibitor. Proc Natl Acad Sci U S A 89: 5635–5639, 1992

15. Cooley J, Takayama TK, Shapiro SD, Schechter NM, Remold-O’Donnell E: The serpin MNEI inhibits elastase-like and chy-motrypsin-like serine proteases through efficient reactions at twoactive sites. Biochemistry 40: 15762–15770, 2001

16. Cooper ME: Pathogenesis, prevention, and treatment of diabeticnephropathy. Lancet 352: 213–219, 1998

17. Asakawa H, Tokunaga K, Kawakami F: Elevation of fibrinogenand thrombin-antithrombin III complex levels of type 2 diabetesmellitus patients with retinopathy and nephropathy. J DiabetesComplications 14: 121–126, 2000

18. Clodi M, Oberbauer R, Bodlaj G, Hofmann J, Maurer G, KostnerK: Urinary excretion of apolipoprotein(a) fragments in type 1diabetes mellitus patients. Metabolism 48: 369–372, 1999

19. Ambuhl P, Amemiya M, Preisig PA, Moe OW, Alpern RJ:Chronic hyperosmolality increases NHE3 activity in OKP cells.J Clin Invest 101: 170–177, 1998

20. Basu TK, Basualdo C: Vitamin A homeostasis and diabetesmellitus. Nutrition 13: 804–806, 1997

21. Rowe DJ, Anthony F, Polak A, Shaw K, Ward CD, Watts GF:Retinol binding protein as a small molecular weight marker ofrenal tubular function in diabetes mellitus. Ann Clin Biochem24[Suppl]:477–482, 1987

22. Tschope C, Reinecke A, Seidl U, Yu M, Gavriluk V, Riester U,Gohlke P, Graf K, Bader M, Hilgenfeldt U, Pesquero JB, Ritz E,Unger T: Functional, biochemical, and molecular investigationsof renal kallikrein-kinin system in diabetic rats. Am J Physiol277: H2333–H2340, 1999

23. Essawy M, Soylemezoglu O, Muchaneta-Kubara EC, ShortlandJ, Brown CB, el Nahas AM: Myofibroblasts and the progressionof diabetic nephropathy. Nephrol Dial Transplant 12: 43–50,1997

24. Sanai T, Sobka T, Johnson T, el Essawy M, Muchaneta-KubaraEC, Ben Gharbia O, el Oldroyd S, Nahas AM: Expression ofcytoskeletal proteins during the course of experimental diabeticnephropathy. Diabetologia 43: 91–100, 2000

25. Sterzel RB, Hartner A, Schlotzer-Schrehardt U, Voit S,Hausknecht B, Doliana R, Colombatti A, Gibson MA, BraghettaP, Bressan GM: Elastic fiber proteins in the glomerular mesan-gium in vivo and in cell culture. Kidney Int 58: 1588–1602, 2000

26. Debelle L, Tamburro AM: Elastin: Molecular description andfunction. Int J Biochem Cell Biol 31: 261–272, 1999

27. Tanno T, Yoshinaga K, Sato T: Alteration of elastin in aorta fromdiabetics. Atherosclerosis 101: 129–134, 1993

28. Kwan CY, Wang RR, Beazley JS, Lee RM: Alterations of elastinand elastase-like activities in aortae of diabetic rats. BiochimBiophys Acta 967: 322–325, 1988

29. Pasquali-Ronchetti I, Baccarani-Contri M: Elastic fiber duringdevelopment and aging. Microsc Res Tech 38: 428–435, 1997

30. Kucich U, Rosenbloom JC, Abrams WR, Bashir MM, Rosen-bloom J: Stabilization of elastin mRNA by TGF-beta: Initial


characterization of signaling pathway. Am J Respir Cell Mol Biol17: 10–16, 1997

31. Geisler N, Plessmann U, Weber K: Amino acid sequence char-acterization of mammalian vimentin, the mesenchymal interme-diate filament protein. FEBS Lett 163: 22–24, 1983

32. Rastaldi MP, Ferrario F, Giardino L, Dell’Antonio G, Grillo C,Grillo P, Strutz F, Muller GA, Colasanti G, D’Amico G: Epithe-lial-mesenchymal transition of tubular epithelial cells in humanrenal biopsies. Kidney Int 62: 137–146, 2002

33. Dalley A, Smith JM, Reilly JT, Neil SM: Investigation of cal-modulin and basic fibroblast growth factor (bFGF) in idiopathicmyelofibrosis: Evidence for a role of extracellular calmodulin infibroblast proliferation. Br J Haematol 93: 856–862, 1996

34. Uchio K, Tuchweber B, Manabe N, Gabbiani G, Rosenbaum J,Desmouliere A: Cellular retinol-binding protein-1 expressionand modulation during in vivo and in vitro myofibroblasticdifferentiation of rat hepatic stellate cells and portal fibroblasts.Lab Invest 82: 619–628, 2002

35. Tooyama I, Sato H, Yasuhara O, Kimura H, Konishi Y, ShenY, Walker DG, Beach TG, Sue LI, Rogers J: Correlation ofthe expression level of C1q mRNA and the number of C1q-positive plaques in the Alzheimer disease temporal cor-tex. Analysis of C1q mrna and its protein using adjacent ornearby sections. Dement Geriatr Cogn Disord 12: 237–242,2001

36. Tong X, Kawabata H, Koeffler HP: Iron deficiency can upregu-late expression of transferrin receptor at both the mRNA andprotein level. Br J Haematol 116: 458–464, 2002

37. Gygi SP, Rochon Y, Franza BR, Aebersold R: Correlation be-tween protein and mRNA abundance in yeast. Mol Cell Biol 19:1720–1730, 1999

38. Ji P, Xuan JW, Onita T, Sakai H, Kanetake H, Gabril MY, SunY, Moussa M, Chin JL: Correlation study showing no concor-dance between EPAS-1/HIF-2� mRNA and protein expressionin transitional cellcancer of the bladder. Urology 61: 851–857,2003


Alterations in the Renal Elastin-Elastase System in...

Documents

Transcript of Alterations in the Renal Elastin-Elastase System in...