Markedly Increased Urinary Preprohaptoglobin and Haptoglobin in Passive Heymann Nephritis: A Differential Proteomics Approach Heidi Hoi-Yee Ngai,† Wai-Hung Sit,† Ping-Ping Jiang,† Visith Thongboonkerd,*,‡,§ and Jennifer Man-Fan Wan*,†,§ Department of Zoology, The University of Hong Kong, HKSAR, People’s Republic of China, and Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine at Siriraj Hospital, Mahidol University, Bangkok, Thailand Received April 29, 2007
Membranous nephropathy (MN), a common cause of idiopathic nephrotic syndrome in adults, remains a potentially devastating problem worldwide. At present, there is no reliable noninvasive method for predicting and/or monitoring this glomerular disease, and its pathophysiology remains poorly understood. In the present study, the urinary proteome profile of rats after 10 days of an induction of passive Heymann nephritis (PHN), which resembles human MN, was compared to that of the baseline (control) urine prior to the induction of PHN by anti-Fx1A injection. Each pool of PHN and control urine samples (n ) 10 each) was labeled with different fluorescent dyes (Cy3 or Cy5), and equal amounts of the labeled proteins of both pools were resolved in the same 2D gel, together with an internal standard labeled with Cy2. Two-dimensional difference gel electrophoresis revealed a number of protein spots whose expression levels were altered during PHN. Eighteen protein spots with >1.5-fold changes and p < 0.05 were selected for subsequent identification by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry. They were successfully identified as serum albumin precursor, R-1antitrypsin, preprohaptoglobin, liver-regeneration-related protein, and transthyretin (which increased during PHN) and E-cadherin, MPP7, tropomyosin β, kallikrein, and R-2u globulin (which decreased in the PHN urine). Among these proteins, the increase in urinary preprohaptoglobin has particularly drawn our attention because of its byproduct, haptoglobin (Hp), which is involved in the protection of tissue damage from hemoglobin-induced oxidative stress. Western blotting and enzyme-linked immunosorbent assay clearly showed a markedly increased level of Hp in the urine, but not in the serum, of the PHN animals. Our findings may lead to a significant advance in the attempt to define a new therapeutic target and/or novel biomarker for human MN. Keywords: preprohaptoglobin • haptoglobin • 2D-DIGE • glomeruli • membranous nephropathy • passive Heymann nephritis • proteomics • urine
Introduction Membranous nephropathy (MN), the most common cause of nephrotic syndrome in adults,1-3 is characterized by subepithelial accumulation of immune complexes. The primary clinical manifestation is proteinuria, and approximately onethird of the patients will develop end-stage renal disease.2,4,5 Up to now, there has been no reliable noninvasive method for predicting and/or monitoring MN, and molecular dissection of MN is still limited. In spite of adequate treatment with available immunosuppressive regimens, therapeutic response is still unsatisfactory, probably because its pathophysiology remains poorly understood. Therefore, better understanding * To whom correspondence should be addressed. (J.M.-F.W.) Phone: 85222990838. Fax: 852-25599114. E-mail:
[email protected]. (V.T.) Phone: 66-2-4184793. Fax: 66-2-4184793. E-mail:
[email protected]. † The University of Hong Kong. ‡ Mahidol University. § These authors made equal contributions as co principal investigators. 10.1021/pr070245b CCC: $37.00
2007 American Chemical Society
of its pathogenic mechanisms and searching for novel therapeutic targets are crucially required to improve the therapeutic outcome. Proteomics, a modern technology to examine a large number of proteins simultaneously, is considered as one of the methods of choice for the aforementioned needs.6,7 When combined with animal models that mimic human MN, proteomic analysis may lead to the discovery of novel biomarkers and new therapeutic targets. The rat model of passive Heymann nephritis (PHN) is a commonly used animal model for the study of human MN.4,8,9 This model is induced in rats by injection with heterologous antibody against a crude autologous tubular antigen, Fx1A.8-10 Similar to the pathogenesis of human MN, injection of antiFx1A induces formation of immune deposits in the lamina rara externa of the glomerular basement membrane (GBM) that cause a membranelike thickening of the capillary wall.8-14 The invasion of the complement C5b-9 membrane-attack complex to the glomerular epithelial cells or podocytes causes massive Journal of Proteome Research 2007, 6, 3313-3320
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research articles production of proteases, reactive oxygen species (ROS), prostanoids, cytokines, TGF-β, and extracellular matrix components, which play a major role in disruption of the functional integrity of the GBM as well as the protein filtration barrier of podocytes with subsequent development of massive proteinuria.4,13,14 In an attempt to unravel the molecular basis of human MN, the present study was conducted using a two-dimensional difference gel electrophoresis (2D-DIGE) method to identify altered urinary proteins during an early stage of the PHN (10 days after the anti-Fx1A injection). Using this technique, a set of proteins whose urinary excretion levels were altered significantly during PHN have been identified. One of these proteins, preprohaptoglobin, has attracted our attention in the present study because of its byproduct, haptoglobin (Hp), which has been implicated in the physiological defense against hemoglobininduced renal toxicity.15,16 Hp is designated as an acute-phase protein that binds to free hemoglobin (Hb) with the highest affinity.17 It has been suggested that Hp crucially prevents glomerular filtration of hemoglobin, hence protecting the kidney against oxidative injury and allowing heme iron recycling, presumably via its antioxidative effect.16,17 Recent studies also showed that Hp polymorphism is associated with diabetic nephropathy18,19 and severe hypertension and proteinuria,20 suggesting its utility as an early noninvasive biomarker and/or new therapeutic target of renal diseases. In the present study, we have demonstrated that not only preprohaptoglobin but also Hp had significantly increased levels in the urine of animals suffering from PHN. Both Western blot and ELISA analyses clearly confirmed that the increased urinary excretion of Hp was exclusive to PHN. The implications of this finding toward the discovery of urinary Hp as a novel early biomarker and/or new therapeutic target for human MN are discussed.
Materials and Methods Rat Model of PHN. The induction of PHN has been performed as described previously.2,8,9,21 Animal care and treatment were conducted according to the guidelines of the Department of Health, the Government of Hong Kong, SAR. In this study, the PHN model was induced in Sprague-Dawley male rats (n ) 10) by intravenous injection of 1.0 mL of anti-Fx1A antibody. Twenty-four hour urine samples were collected on day 0 (before the anti-Fx1A injection to serve as basal controls) and on day 10 (10 days after the anti-Fx1A injection) for subsequent measurements of urine albumin and renal creatinine clearance. For proteomic analysis, the urine samples were collected within a 4 h period when the rats were housed in individual metabolic cages and received free water, but without food to prevent contamination of proteins from food particles.22,23 On day 10, after the completion of urine collection, the animals were sacrificed and their sera were collected. The kidneys were also harvested for renal histopathological and immunofluorescence examinations. Measurements of 24 h Urine Albumin, Serum Creatinine, and Renal Creatinine Clearance. Urinary albumin was measured using a commercial kit according to the manufacturer’s instructions (BioSystems, Barcelona, Spain). Serum and urinary creatinine levels were analyzed by a standard spectrophotometric protocol. Briefly, the samples were added into picric acid and sodium hydroxide containing SDS. Then the samples were allowed to stand at room temperature for 10 min, and the absorbance was measured by a UV-vis spectrophotometer at 505 nm. Creatinine clearance was calculated using the following 3314
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Ngai et al. Table 1. Experimental Design of 2D-DIGE Gels and Fluorophore Labeling Schemea
Cy2 Cy3 Cy5
gel 1
gel 2
internal standard control (baseline pool) disease (PHN pool)
internal standard disease (PHN pool) control (baseline pool)
a Samples of 50 µg of urinary proteins from the baseline control and PHN pools were labeled with Cy3 or Cy5 dye, respectively (or vice versa). A 50 µg sample of the internal standard containing equal amounts of each sample (25 µg from the baseline control and 25 µg from PHN urine) was labeled with Cy2. These three samples (control, PHN, and internal standard), which were labeled with three different fluorescence dyes, were then resolved simultaneously within the same 2D gel.
formula: creatinine clearance (mL/min) ) [urine creatinine concentration (mg/dL) × 24 h urine volume (mL)]/[serum creatinine concentration (mg/dL) × 1440 (min)]. 2D-DIGE Analysis. We investigated changes in the urinary proteome that could represent potential biomarkers for MN by comparing the urine collected from the animals on day 0 (healthy self-controls, before the induction of PHN) with that of day 10 (PHN). Immediately after the collection, the urine samples were centrifuged at 1000g for 5 min. After removal of cell debris and nuclei, proteins in the supernatants were precipitated with 20% trichloroacetic acid and acetone containing 20 mM dithiothreitol (DTT) at -20 °C overnight followed by three washes with acetone/20 mM DTT. The pellets centrifuged at 12000g for 5 min were resuspended in a buffer containing 7.92 M urea, 0.06% SDS, 120 mM DTT, 3.2% Triton X-100, 22.4 mM Tris-HCl, and 17.6 mM Tris base (pH 8.0). The protein concentration of each sample was measured by spectrophotometry using a Bio-Rad protein microassay, based on Bradford’s method. Equal amounts of total protein derived from individual urine samples collected at the same time point (day 0 or 10) were mixed and used as the pooled urine sample for subsequent 2D-DIGE analysis. A total of 2 2D-DIGE gels (each containing the proteomes of pooled PHN urine from 10 individual animals and pooled control urine from 10 individual animals) were run. In each gel, 50 µg of proteins from pooled control and pooled PHN samples were incubated with 400 pmol of Cy3 and Cy5, respectively, and vice versa, on ice for 30 min. Also, 50 µg of an internal standard comprising equal amounts of control and PHN proteins labeled with Cy2 was added to each gel. The labeling reaction was quenched by the addition of 1 mL of 10 mM lysine and subsequently incubated on ice for 15 min. Therefore, three pooled samples (control, PHN, and internal standard), which were labeled with three different fluorescence dyes, were simultaneously resolved in each 2D-DIGE gel (Table 1). After differential labeling, the mixture of pooled samples and rehydration buffer (containing 8 M urea, 4% CHAPS, 1% IPG buffer, and 13 mM DTT) was rehydrated onto 18 cm, pH 3-10 (linear), immobilized pH gradient (IPG) strips using IPGphor II (GE Healthcare, Uppsala, Sweden) at 50 V for 12 h. Isoelectric focusing was then performed at 500 V for 1 h, 1000 V for 1 h, and finally 8000 V for 12 h. After the completion of isoelectric focusing (first dimensional separation), the proteins on the IPG strips were then incubated for 15 min in an equilibration buffer containing 6 M urea, 2% SDS, 20% glycerol, 13 mM DTT, and 0.375 M Tris/HCl (pH 8.8). The IPG strips were further equilibrated in a similar equilibration buffer, in which DTT was replaced with 2.5% iodoacetamide. The second dimensional separation was performed using a 12.5% acrylamide slab gel
research articles
Increased Urinary Preprohaptoglobin and Hp in PHN
and run with 30 mA/gel for 30 min followed by 60 mA/gel until the dye front reached the bottom of the gel. Image Acquisition and Analysis. Gel images were scanned on a DIGE-enabled Typhoon 9400 variable-mode imager (GE Healthcare) at an excitation wavelength of 670 nm/30 nm (maximum/bandwidth) for Cy5 (laser 633-red) or 580 nm/30 nm for Cy3 (laser 532-green) labeled samples. The images were processed with DeCyder software version 5.01 (GE Healthcare) to identify changes in the spot fluorescence intensities. The 2D gel images of the PHN urine proteome were compared to those of the control urine proteome, using the internal standard for standardization of the quantitation. The differential analysis was performed for each of the two gels in parallel using the DIA module of DeCyder with a value of 1000 as the initial estimate of the protein spots present. The DIA analysis allows direct comparisons of the intensities of individual protein spots between the two different samples within the same gel (in our case, PHN versus control urine). These DIA analyses were collated into a single analysis using the BVA module of DeCyder, and final expression ratios (PHN/control) of individual protein spots were determined. Only spots with >1.5fold changes and p < 0.05 in their intensity volumes after normalization were reported as differentially expressed proteins and subjected to subsequent identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) followed by peptide mass fingerprinting. MALDI-TOF MS and Peptide Mass Fingerprinting. In-gel tryptic digestion was performed as described previously.21 Protein identification was performed using a reflectron MALDITOF mass spectrometer (Autoflex, Bruker Daltonics, Leipzig, Germany). Peptide mass fingerprinting of the peptide fragments was performed with the ProFound search engine (http:// prowl.rockefeller.edu/prowl-cgi/profound.exe) and the Mascot software (http://www/matrixscience.com). Haptoglobin Detection by Western Blot Analysis. In Western blot analysis, the precipitated urine proteins and the serum samples were mixed with a 2× standard Laemmli buffer (1:1, v/v) and heated at 95 °C for 5 min. A 20 µg sample of total protein obtained from each animal was loaded in each lane of the SDS-PAGE gels. Two duplicated gels were run simultaneously; one was stained with Coomassie Brilliant Blue R-250, whereas the other one was subjected to Western blot analysis. For serum samples, proteins were also resolved by twodimensional gel electrophoresis. Resolved proteins in SDSPAGE or the 2D gel for immunoblotting were transferred onto a PVDF membrane and probed with polyclonal anti-haptoglobin (Abcam, Cambridge, U.K.) followed by peroxidase-conjugated secondary antibody. The immunoreactive bands were visualized using enhanced chemiluminescence substrate (GE Healthcare) and autoradiography. The intensity of the haptoglobin immunoreactive band (at approximately 40 kDa) was measured by densitometric analysis using Quantity One (BioRad, Hercules, CA). Haptoglobin Detection by ELISA. The urinary haptoglobin level was also measured by an enzyme-linked immunosorbent assay (ELISA) kit. The test was performed according to the manufacturer’s instructions (Immunology Consultants Lab Inc., Newberg, OR). Statistical Analyses. All data are expressed as the mean ( SEM. Differences between groups were analyzed for statistical significance by analysis of variance and the t-test using SPSS software (version 11.5). A p value of 1.5-fold changes and p < 0.05 were selected for subsequent identification with MALDI-TOF MS followed by peptide mass fingerprinting. Table 2 shows the identities of these altered proteins. The spot IDs correspond to those labeled in Figure 3 (“D” indicates the down-regulated spots, whereas “U” represents the up-regulated ones). The proteins with increased urinary excretion levels during PHN included serum albumin precursor, R-1-antitrypsin, preprohaptoglobin, liverregeneration-related protein, and transthyretin, whereas the proteins whose urinary excretion declined during PHN included E-cadherin, MPP7, tropomyosin β, kallikrein, and R-2u globulin. Some of the altered protein spots were identified as the same protein, most likely due to post-translational modifications, which are common for proteins present in human body fluids.23,24 Markedly Increased Haptoglobin Level in the Urine, Not in the Plasma, of PHN. Five protein isoforms (with varying isoelectric points ranging from approximately 5.7 to 7.2 and with modest changes in molecular masses) which were consistently overexpressed in the urine of PHN rats (Figure 4) were identified as preprohaptoglobin (NCBI ID gi|204657, accession number AAA41349). The magnitude of this increase was varied from 1.5- to 3.0-fold for each isoform. This finding was not observed in adriamycin-induced nephropathy, a model of nonMN nephropathy, which had a normal level of urinary preprohaptoglobin (data not shown). Preprohaptoglobin is a primary translation product of Hp, which exists in three major phenotypic forms designated as Hp1-1, Hp2-1, and Hp2-2 in human body fluids.17 It was next of interest to confirm that Hp also increased in the PHN urine and to examine whether the increase in Hp is specific only in the urine. Western blot analysis showed that Hp was detectable as a 40 kDa immunoreactive band with a markedly increased level (approximately 24-fold) in the PHN urine of five individual rats while the band of Hp was barely detected in the control urine (Figure 5). ELISA measurement also confirms the proteomic and immunoblotting data (Figure 6). In contrast to the urine results, Hp was detectable in both basal control and PHN sera, with comparable levels analyzed by 1D and 2D Western blotting (parts A and B, respectively, of Figure 7). 3316
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Satisfactory therapeutic response and prognosis of human MN are related to its early detection. Renal biopsy, while it provides a specific diagnosis, is an invasive procedure with considerably significant complications, particularly in patients with bleeding tendency or skin infection. Thus, a renal biopsy may not be possible or my be contraindicated in certain highrisk patients. Searching for noninvasive biomarkers is therefore the hallmark for detection and prediction of the treatment outcome of human MN. Urine is an ideal source of materials to search for potential disease-related biomarkers as it bathes the affected tissue (the kidney) and can be obtained easily by noninvasive methods.22,23 In the present study, we employed a PHN rat model to evaluate whether there is a set of urinary proteins that can be used as the novel biomarkers for PHN and/or human MN. Clinical characteristics of the PHN rats after 10 days of the antiFx1A injection mimicked those of human MN. These included profound proteinuria, mesangial expansion, thickened GBM, and granular deposition of IgG within the glomeruli. However, renal function (as determined by renal creatinine clearance) of the PHN animals 10 days after the anti-Fx1A injection remained normal. Hence, this animal model at the mentioned time point is suitable for such a purpose to define novel biomarkers for early disease detection. Moreover, an extensive study of urinary proteins in this animal model may also lead to a better understanding of the disease pathophysiology in human MN. Our aim was not to examine the proteome profile at the more advanced stage when the renal function declined, as changes in the late stage may be the result of severe kidney damage such as glomerulosclerosis and tubulointerstitial injury. The 2D-DIGE proteomic technique was applied to examine changes in the urinary proteome profile of PHN rats. To our knowledge, this is the first study that evaluates the urinary proteome of PHN or MN using the 2D-DIGE approach. The advantages of this approach are the high reproducibility (as there is an internal standard used in the system), the absence of gel-to-gel variability (as the two sets of different samples are simultaneously resolved within the same gel), and the higher sensitivity when compared to other gel-based techniques.25 Using an appropriate analytical software and statistics, quantitative intensity analysis revealed a number of differentially expressed protein spots between the PHN urine and the basal control urine. Some of the proteins, identified by MALDITOF MS and peptide mass fingerprinting, may be helpful for better understanding the pathogenic mechanisms or pathophysiology of MN, whereas some may potentially serve as novel biomarkers for human MN. For those proteins whose excretion levels were decreased in the PHN urine, E-cadherin markedly decreased by approximately 42-fold (Table 2). It is a type I membrane, calciumdependent, cell adhesion molecule that plays an important role in maintaining the glomerular permeability.26,27 A marked decrease in cadherin, therefore, is expected to be associated with the impaired glomerular permselectivity, which leads to proteinuria and ultimately declined renal function. Kallikrein is a serine proteinase that is involved in production of the potent vasodilator bradykinin.28 We identified a marked reduction of two isoforms of kallikrein in the PHN urine (62- and 83-fold decreases, respectively) (Table 2). Its reduction might be associated with hypertension, a common feature found in MN. MPP7 (M-phase phosphoprotein 7) was reduced by
Increased Urinary Preprohaptoglobin and Hp in PHN
research articles
Figure 3. 2D-DIGE analysis of the urinary proteome. (A) The pooled control urinary proteins (n ) 10 rats) were labeled with Cy3 (green), whereas the pooled PHN urinary proteins (n ) 10 rats) were labeled with Cy5 (red). (B) The pooled control urine sample was labeled with Cy5 (red), whereas the pooled PHN urine sample was labeled with Cy3 (green). In each gel, equal amounts of total protein from either pool (50 µg each) were mixed, and both pools were simultaneously run within the same 2D gel with an internal standard labeled with Cy2. 2D-PAGE was performed using a linear pH gradient 3-10 IPG strip and a 12.5% acrylamide second dimensional gel. Quantitative intensity analysis using DeCyder software and statistics revealed alterations in the expression levels of a number of protein spots. “D” indicates the down-regulated spots, whereas “U” represents the up-regulated spots. These spot IDs correspond to those shown in Table 2.
Figure 4. 2D-DIGE zoom-in images of the increased preprohaptoglobin excretion in the PHN urine. (A) 3D view of the preprohaptoglobin spots (U5-U9) (the left image is from the control urine, whereas the right image is from the PHN urine). (B) Cy3 image view (the left panel represents the control urine, whereas the right panel is from the PHN urine). (C) Cy5 image view (the left panel represents the control urine, whereas the right panel is from the PHN urine). (D) Combined Cy3 and Cy5 image view of the data shown in (B) and (C). The white arrows indicate each of the preprohaptoglobin spots.
approximately 75-fold in the PHN urine (Table 2). While its major function is defined as a chromatin assembly factor thought to mediate chromatin assembly in DNA replication and DNA repair,29 its role in nephrotic syndrome remains unclear. Evaluation of its functional significance in the kidney deserves further investigation, which may lead to better understanding of the pathophysiology of PHN and human MN.
Interestingly, we identified an increase in excretion of five isoforms of urinary preprohaptoglobin during PHN. Varying isoelectric points and modest changes in the molecular masses of the five isoforms of preprohaptoglobin were most likely due to post-translational modifications (probably glycosylation). This finding was not observed in adriamycin-induced nephropathy, a non-MN model, which had normal levels of urinary Journal of Proteome Research • Vol. 6, No. 8, 2007 3317
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Ngai et al.
Table 2. Summary of the Significantly Altered Proteins in the PHN Urine as Compared to the Basal Control Urine spot no.a
D1 D2 D3 D4 D5 D6 U1 U2 U3 U4 U5 U6 U7 U8 U9 U10 U11
U12
GenInfo IDb
protein name
epithelial-cadherin precursor (E-cadherin) (uvomorulin) Mpp7 protein [Mus musculus] tropomyosin β chain (tropomyosin 2) (β-tropomyosin) kallikrein 7, kallikrein 1 [Rattus norvegicus] kallikrein 7, kallikrein 1 [R. norvegicus] R-2u globulin serum albumin precursor [contains neurotensin-related peptide (NRP)] serum albumin precursor (contains NRP) R-1-antitrypsin precursor R-1-antiproteinase precursor (R-1-antitrypsin) (R-1-proteinase inhibitor) preprohaptoglobin preprohaptoglobin preprohaptoglobin preprohaptoglobin preprohaptoglobin liver-regeneration-related protein LRRG03 [R. norvegicus] serine protease inhibitor R-1, R-1-antitrypsin, R-1-antitrypsin (protease inhibitor), serine (or cysteine) proteinase inhibitor clade A chain D, rat transthyretin
a
accession no.
cov (%)
pIc
Mwc
Z score
MOWSE score
p value (t test)
av ratio of changesd
gi|13431333
Q9R0T4
14
4.7
99.21
2.33
44
0.0014
-42.46
gi|29437038 gi|20178269
AAH49662 P58775
57 61
6.6 4.7
40.70 32.93
2.43 2.43
66 45
0.0025 0.0130
-75.66 -36.80
gi|6981132 gi|6981132 gi|204264 gi|113580
NP_036725 NP_036725 AAA41199 P02770
29 29 62 34
5.6 5.6 5.4
29.52 29.52 17.34
2.21 1.28 2.38
60 42 92
0.0014 0.0024 0.0060
-82.80 -62.32 -16.75
gi|113580 gi|203063 gi|112889
P02770 AAA40788 P17475
34 36 35
6.1 5.7 5.7
70.70 45.99 46.29
2.42 2.43 2.43
34 44 37
0.0075 0.0160 0.0190
1.61 3.94 2.21
gi|204657 gi|204657 gi|204657 gi|204657 gi|204657 gi|33187764
AAA41349 AAA41349 AAA41349 AAA41349 AAA41349 AAP97736
32 33 33 38 36 51
7.2 7.2 7.2 7.2 7.2 7.3
30.43 30.43 30.43 30.43 30.43 79.00
2.15 2.22 2.33 2.25 2.40 2.43
76 89 54 81 90 132
0.0200 0.0071 0.0130 0.0200 0.0480 0.0058
1.69 2.57 3.02 1.55 1.92 4.48
gi|51036655
NP_071964
25
5.7
46.28
2.17
74
0.0170
4.83
gi|3212535
1GKE
73
6
13.11
2.43
97
0.0270
2.55
b
c
d
The numbers correspond to those shown in Figure 3. According to the NCBI database. Theoretical pI and molecular mass (kDa). A positive value signifies an increased excretion level whereas a negative value signifies a decreased excretion level in terms of fold differences.
Figure 6. ELISA measurement of urinary Hp. The urinary Hp level was measured by the ELISA method. Hp in the PHN urine significantly increased. The asterisk indicates p < 0.01 (n ) 10 for each group).
Figure 5. Western blot analysis of urinary Hp. Equal amounts of total protein (20 µg) isolated from the urine of each rat were loaded into each lane (n ) 5 in each group). The proteins were resolved with 12% SDS-PAGE. (A) The Hp band (at approximately 40 kDa) was clearly detected in all the PHN urine samples, but was almost undetectable in all the controls. (B) The band intensities were quantitated and are presented as intensity volumes (vol %). The asterisk indicates p < 0.01.
preprohaptoglobin (data not shown), indicating that the increased urinary preprohaptoglobin might be specific for PHN. However, other models of nephropathy should also be evaluated to strengthen our data. Preprohaptoglobin (totally 272 amino acid residues in rats) is a primary translation product of Hp (totally 347 amino acid residues in rats), which contains R and β subunits.30,31 Preprohaptoglobin can be N-glycosylated and dimerized prohapto3318
Journal of Proteome Research • Vol. 6, No. 8, 2007
globin post-translationally cleaved to produce the native tetrameric structure linked by intermolecular disulfide bonds.32 A single arginine residue is found at the R-β subunit junction of prohaptoglobin. The alignment of these two subunits with serine protease precursors shows 75% homology for the R subunit and 90% for the β subunit and suggests that posttranslational processing of prohaptoglobin involves cleavage of the Arg-Ile bond and extraction of the Arg residue and that both subunits contribute to haptoglobin’s biological activity.32 Hp binds with Hb with the highest affinity and prevents renal iron load and subsequent oxidative damage mediated by free Hb.15-17 The Hb-Hp complex is metabolized in the hepatic system. Biosynthesis of Hp has long been known to occur in the liver. However, other locales of tissue Hp have been observed in adipose tissue, skin, lung, and kidney, but with smaller amounts as compared to its expression in the liver.33,34 The elevated serum Hp level has been reported in patients with glomerular diseases, including glomerulonephritis, MN, and IgA nephropathy.35 In contrast, the declined serum level of Hprelated protein precursor has been reported in type 2 diabetes
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Increased Urinary Preprohaptoglobin and Hp in PHN
Figure 7. Western blot analysis of serum Hp. The serum Hp level was evaluated in both baseline control (day 0) and PHN (day 10) rats by 1D Western blot analysis (A). The data showed comparable levels of serum Hp in the two groups. 2D Western blot analysis revealed expression of Hp in the serum as four major isoforms (B).
patients with microalbuminuria.36,37 However, the Hp level in the urine had not previously been examined in human glomerular diseases. We report herein, for the first time, the marked increase in urinary excretion of Hp during PHN or MN. However, it is still unclear how Hp contributes to the pathogenic mechanisms of PHN. The pathogenesis of MN involves in situ formation of subepithelial immune deposits that cause glomerular injury by damaging podocytes through the activation of complement C5b-9.4,5,14 Recent evidence shows that prohaptoglobin is proteolytically cleaved by the complement C1r-like protein.38 We, therefore, hypothesize that Hp is involved in the complement-dependent podocyte damage. Complement (C5b-9) activation leads to release of proteases, oxidants, prostanoids, and a few growth factors,3-5,39 which in turn destroy the functional integrity of the GBM. Accumulation of injurious reactive oxygen species produced by activated neutrophils and macrophages can also cause renal tissue damage by initiating local lipid peroxidation and cause alteration in glomerular permeability.14 The podocyte membrane protein such as megalin, which is the target antigen of the nephritogenic antibodies, easily undergoes autoxidation.40 Hp might reduce loss of Hb through the glomeruli, allowing heme iron recycling and hence protecting the kidney against oxidative stress induced by free Hb as well as by local lipid peroxidation. The evidence of increased susceptibility of Hb-driven lipid oxidation, demonstrated in conditions of hypohaptoglobinemia or anhaptoglobinemia in humans and in Hp-deficient mice, indirectly supports this hypothesis.16 Hp is also considered as an anti-inflammatory agent, as well as an immunomodulator. It has an inhibitory effect on prostaglandin synthesis,41 an ability to dampen the metabolism and function of neutrophils, monocytes, and macrophages,42 and a capability to interfere with T- and B-cell proliferation.43 Hp binds to a variety of immune cell types, including monocytes, granulocytes, natural killer cells, and subsets of T and B lymphocytes,44 and can act as a potent immunosuppressor or immunomodulator of lymphocyte (particularly helper T-cell) function.45 Hp has also been demonstrated to inhibit the inappropriate self-association of damaged (or misfolded) extracellular proteins during renal injury via its chaperone action.46
In summary, we present the first evidence that urinary excretion levels of preprohaptoglobin and Hp are markedly elevated in an early stage of rat PHN. The increase in their excretion levels may be a compensatory mechanism to prevent further renal injury from the oxidative stress occurring during PHN. Several lines of evidence gleaned from our present study, at least, prompt us to believe that preprohaptoglobin and Hp can possibly be developed as novel urine biomarkers for MN as their increased urinary excretion levels were exclusive to PHN. Additionally, this striking finding was specific only in the urine, not in the serum. Moreover, the markedly increased urine preprohaptoglobin and Hp levels can be detected at an early stage of the disease, and the detection procedures are noninvasive. We, therefore, suggest that urinary preprohaptoglobin and Hp may potentially be useful as novel biomarkers for MN. Further study should be conducted to evaluate urinary preprohaptoglobin and Hp levels in human MN. Abbreviations: 2D-DIGE, two-dimensional difference gel electrophoresis; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; GBM, glomerular basement membrane; Hp, haptoglobin; IPG, immobilized pH gradient; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MN, membranous nephropathy; PHN, passive Heymann nephritis.
Acknowledgment. This study was supported by the Hong Kong Healthcare Association, Ltd., Hong Kong, and the Matching Grant Scheme under the University Grants Committee of the Hong Kong Special Administration Region, China (Project No. 20600305) (to J.M.-F.W.) and by Siriraj Grant for Research and Development, Mahidol University, and Vejdusit Foundation, Thailand Research Fund, Commission on Higher Education, National Center for Genetic Engineering and Biotechnology, and National Research Council of Thailand (to V.T.). References (1) Wasserstein, A. G. Membranous glomerulonephritis. J. Am. Soc. Nephrol. 1997, 8, 664-674. (2) Ronco, P.; Debiec, H. Molecular pathomechanisms of membranous nephropathy: from Heymann nephritis to alloimmunization. J. Am. Soc. Nephrol. 2005, 16, 1205-1213. (3) Glassock, R. J. Diagnosis and natural course of membranous nephropathy. Semin. Nephrol. 2003, 23, 324-332. (4) Couser, W. G. Membranous nephropathy: a long road but well traveled. J. Am. Soc. Nephrol. 2005, 16, 1184-1187. (5) Nangaku, M.; Shankland, S. J.; Couser, W. G. Cellular response to injury in membranous nephropathy. J. Am. Soc. Nephrol. 2005, 16, 1195-1204. (6) Mann, M.; Hendrickson, R. C.; Pandey, A. Analysis of proteins and proteomes by mass spectrometry. Annu. Rev. Biochem. 2001, 70, 437-473. (7) Aebersold, R.; Mann, M. Mass spectrometry-based proteomics. Nature 2003, 422, 198-207. (8) Heymann, W.; Hackel, D. B.; Harwood, S.; Wilson, S. G.; Hunter, J. L. Production of nephrotic syndrome in rats by Freund’s adjuvants and rat kidney suspensions. 1951. J. Am. Soc. Nephrol. 2000, 11, 183-188. (9) Cybulsky, A. V.; Quigg, R. J.; Salant, D. J. Experimental membranous nephropathy redux. Am. J. Physiol. Renal Physiol. 2005, 289, F660-F671. (10) Kerjaschki, D.; Ullrich, R.; Exner, M.; Orlando, R. A.; Farquhar, M. G. Induction of passive Heymann nephritis with antibodies specific for a synthetic peptide derived from the receptorassociated protein. J. Exp. Med. 1996, 183, 2007-2015. (11) Kerjaschki, D.; Neale, T. J. Molecular mechanisms of glomerular injury in rat experimental membranous nephropathy (Heymann nephritis). J. Am. Soc. Nephrol. 1996, 7, 2518-2526. (12) Salant, D. J.; Cybulsky, A. V. Experimental glomerulonephritis. Methods Enzymol. 1988, 162, 421-461.
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