Original Article doi: 10.1111/joim.12214
Excessive activation of the alternative complement pathway in autosomal dominant polycystic kidney disease Z. Su1,*,†, X. Wang1,*, X. Gao1,*, Y. Liu2, C. Pan3, H. Hu1, R. P. Beyer4, M. Shi3, J. Zhou5, J. Zhang3, A. L. Serra2,6, R. P. W€uthrich2,6 & C. Mei1 From the 1Kidney Institute, Department of Nephrology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China; 2Institute of Physiology, University of Zurich, Zurich, Switzerland; 3Division of Neuropathology, Harborview Medical Center, University of Washington School of Medicine; 4Department of Environmental & Occupational Health Sciences, University of Washington, Seattle, WA; 5 Harvard Institutes of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; 6Division of Nephrology, University Hospital Zurich, Zurich, Switzerland †Present address: Division of Nephrology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, Zhejiang, China
Abstract. Su Z, Wang X, Gao X, Liu Y, Pan C, Hu H, Beyer RP, Shi M, Zhou J, Zhang J, Serra AL, W€ uthrich RP, Mei C (Second Military Medical University, Shanghai; University of Zurich, Zurich; University of Washington School of Medicine, Seattle, WA; University of Washington, Seattle, WA; Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; University Hospital Zurich, Zurich). Excessive activation of the alternative complement pathway in autosomal dominant polycystic kidney disease. J Intern Med 2014; 276: 470–485. Objectives. The complement system is involved in many immune complex-mediated kidney diseases, yet its role in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) has not been examined in detail. Methods and Results. Screening of the glycoproteome of urine samples from ADPKD patients revealed that levels of complement factor B (CFB), serpin peptidase inhibitor, complement component 1 inhibitor (SERPING1) and complement component 9 (C9) increased, whereas complement component 1, r subcomponent-like (C1RL), CD55 and CD59 levels decreased with disease progression. Immunostaining and Western blot analysis confirmed the enhanced expression of CFB and C9 in cystic
Introduction Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development and growth of innumerable cysts that originate from the tubular epithelium of nephrons [1]. Mutations in the PKD1 or PKD2 genes are the primary cause of ADPKD [1, 2], resulting in disturbances of multiple *These authors contributed equally to this paper.
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kidneys from ADPKD patients. Immunostaining also showed that the expressions of CFB and C9 in renal biopsy tissues from patients with other types of chronic kidney disease were lower than in tissues from ADPKD patients. The effect of the complement inhibitor rosmarinic acid (RMA) was evaluated in Pkd1 / mice and Han:SPRD Cy/+ rats. Compared with vehicle-treated Pkd1 / animals, RMA-treated mice had significantly lower serum creatinine ( 50%) and blood urea nitrogen ( 78%) levels, two kidneys/body weight ratio ( 60%) and renal cystic index ( 60%). Similar results were found in Cy/+ rats. Lower numbers of Ki67-positive nuclei and inflammatory cells and reduced fibrosis were observed in both animal models upon treatment with RMA. Conclusions. These results suggest that excessive activation of the alternative complement pathway is associated with ADPKD progression, probably mediated by cyst-lining epithelial cell proliferation, tubulointerstitial inflammatory cell infiltration and fibrosis. Targeting the complement system might represent a new therapeutic strategy for ADPKD. Keywords: autosomal dominant polycystic kidney disease, complement, complement inhibitor, glycoproteomics, urine.
cellular signalling pathways. The pathophysiological changes in the disease consist of cell proliferation and apoptosis, fluid secretion, altered cellular polarity and interstitial fibrosis, leading to renal failure in 50% of patients by the age of 60 [3, 4]. The complement system is involved in many types of immune complex-mediated kidney disease, such as IgA nephropathy (IgAN), lupus nephritis (LN)
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and membranous and membranoproliferative nephropathy, yet its role in the pathogenesis of ADPKD is unknown. The complement system can be activated via three different pathways: the classical, alternative and mannose-binding lectin (MBL) pathways. Although essential to defend against pathogens, excessive activation of the complement system causes inflammation and tissue injury. Recently, using microarray analysis, Burtey et al. [5] found that nine different complement component genes were over-expressed in the kidneys of Han:SPRD rats, which is a model for inherited polycystic kidney disease. Furthermore, Lai et al. [6] identified seven complement proteins in the cystic fluid from patients with ADPKD. The purpose of the present study was to examine the activity of the complement system as a function of ADPKD progression and to explore the possibility that this system could provide a novel therapeutic target for ADPKD. Methods Study participants Ten healthy adults served as control subjects. Thirty patients with ADPKD were recruited from Shanghai Changzheng Hospital. The diagnostic criteria for ADPKD included a positive family history of ADPKD and characteristic findings on B-ultrasonography [7] as well as the absence of primary disease of another system; in addition, denaturing high-performance liquid chromatography was used to reveal a PKD1 mutation. Based on the chronic kidney disease (CKD) stages, patients were classified as ADPKD stage 1, ADPKD stage 2–3 and ADPKD stage 4–5 (with 10 patients per group). These four groups (including the control group) were comparable with regard to age and gender (Table 1). Informed consent was obtained from all participants. Human urine collection For collection of the second-morning urine samples, subjects in the four groups fasted (food and fluid) from 22:00 on the previous day until the second void on the study morning. Fresh mid-stream urine was centrifuged, and the supernatant was collected and immediately stored at 80 °C. Protease inhibitors were not used in the urine collection.
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and centrifuged at 4 °C. The supernatant was collected and stored at 80 °C. Analysis of the urinary glycoproteome Total protein was extracted using acetone precipitation from 10 mL urine from each subject. An equal amount of protein from the 10 individual samples in each group was combined, and 120 lg protein from each group was digested with trypsin in parallel and then labelled with isobaric tag for relative and absolute quantification (iTRAQ) reagents (Applied Biosystems, Foster City, CA, USA) as described previously [8]. The first experiment used 4-Plex iTRAQ, with reagents 114, 115, 116 and 117 added to the control, ADPKD stage 1, ADPKD stage 2–3 and ADPKD stage 4–5 groups, respectively. To reduce experimental and intergroup deviations, a second experiment was performed using 8-Plex iTRAQ, with reagents 113, 114, 115 and 116 added to the control, ADPKD stage 1, ADPKD stage 2–3 and ADPKD stage 4–5 groups, respectively, as a repeat experiment reagents 117, 118, 119 and 121 were added to the control, ADPKD stage 1, ADPKD stage 2–3 and ADPKD stage 4–5 respective groups, which as the second repeat experiment. The iTRAQlabelled digests were combined, loaded onto a C18 column (Waters, Milford, MA, USA) to desalt, and were then oxidized with 10 mmol L 1 sodium periodate at room temperature for 1 h. After a second desalting step in a C18 column, the samples were incubated with affinity gel hydrochlorothiazide resin (Bio-Rad Laboratories, Berkeley, CA, USA) at room temperature for 24 h, washed and reacted with peptide-N-glycosidase F (PNGase F; New England BioLabs, Beverly, MA, USA) at 37 °C for 48 h. The supernatant was collected, and the specimens were dissolved in 0.5% trifluoroacetic acid (TFA) and separated with reverse-phase chromatography. Mass spectrometry (MS) was performed with a 4800-matrix-assisted laser desorption ionization time of flight tandem mass spectrometer (MALDITOF/TOF; Applied Biosystems). The MS spectra were searched against the international protein index human protein database (version 3.18) using the Proteinpilot 2.0 software (Applied Biosystems) for peptide and protein identification and quantification. Western blot analysis
Human blood sample collection For the serum C3 and C4 assays, fasting venous blood (5 mL) was collected in the early morning
Urine concentrations of complement factor B (CFB), complement component 1, r subcomponent-like (C1RL), serpin peptidase inhibitor, complement ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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Table 1 Clinical characteristics of ADPKD patients and control subjects
Sex M/F) Age (years) 24-h urine protein (mg) SCr (lmol L
1
)
Control
ADPKD stage 1
ADPKD stage 2–3
ADPKD stage 4–5
6/4
6/4
6/4
6/4
48 3
44 5
60 14
67 16
79 26
91 31
62 11
66 16
160 20*
445 30*
58 15*
16 8*
1
)
1.1 0.4
1.1 0.2
1.2 0.2
1.0 0.3
Serum C4(g L
1
)
0.2 0.1
0.2 0.1
0.3 0.1
0.3 0.1
1
per 1.73 m2)
123 17
50 4
Serum C3(g L
eGFR (mL min
130 6
46 5
The data shown are the mean SD. ADPKD, autosomal dominant polycystic kidney disease; SCr, serum creatinine; eGFR, estimated glomerular filtration rate using modification of diet in renal disease (MDRD) formula. *P < 0.05 compared with control. There were no significant differences in serum C3 and C4 levels between the four groups.
component 1 inhibitor (SERPING1), CD55, CD59 and C9 were measured by Western blot analysis. In brief, an equal amount of total protein from each group was collected, and the glycoproteins were enriched as described above. Glycoproteins (20 lg per group) were separated by 8–16% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride membranes. The membranes were blocked and probed overnight at 4 °C with 1 : 1000 goat anti-human CFB (R&D systems, Minneapolis, MN, USA), 1 : 350 rabbit anti-human C1RL (SigmaAldrich, St. Louis, MO, USA), 1 : 200 mouse antihuman SERPING1 (Abnova Corporation), 1 : 1000 mouse anti-human C9 (Abcam, Cambridge, UK), 1 : 200 mouse anti-human CD55 (Abcam) or 1 : 500 mouse anti-human CD59 (Novus Biologicals) antibodies, followed by peroxidase-conjugated anti-goat, anti-mouse or anti-rabbit IgG (1 : 2000) at room temperature for 1 h. An enhanced chemiluminescence (Amersham Pharmacia) was used for protein band visualization. CFB and C9 in the human and rat kidney tissues were also detected by western blotting as described above, using 1 : 1000 goat anti-human CFB or 1 : 1000 mouse antihuman C9 antibodies. Normal human renal tissues were obtained from the uninvolved pole of kidneys that were removed due to a localized cancer. Autosomal dominant polycystic kidney disease renal tissues were obtained from patients who required nephrectomy of one or both cystic kidney (s) to better accommodate a renal allograft. In-gel digestion and MS validation Enriched urinary glycoproteins from each group were separated by SDS-PAGE, fixed and stained with Coomassie blue. The protein bands to be 472
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identified (CFB, C1RL, SERPING1, CD55, CD59 and C9) were excised from the gel, cut into 1-mm3 pieces and incubated with 50 lL bleaching solution (50% acetonitrile and 25 mmol L 1 bicarbonate) at room temperature for 30 min. After removing the bleaching solution, the above procedures were repeated until the gel became transparent. The gel was then dried in a vacuum centrifuge to obtain white granules, to which 1 mL trypsin (20 ng lL 1 dissolved in 20 mmol L 1 bicarbonate) was added for overnight digestion at 30 °C. The peptide was obtained after washing and desalting and then dried and re-dissolved in 2 lL 0.2% TFA solution for MS identification (MALDI-TOF/TOF) as previously described [9]. Immunoturbidity of serum C3 and C4 Serum C3 and C4 levels in ADPKD patients were detected by immunoturbidity using C3 and C4 assay kits, respectively, according to the manufacturer’s instructions (Dade Behring, Newark, NJ, USA). Conditional Pkd1
/
mice
Conditional Pkd1 / mice [10] were a gift from Professor G. Germino, and T. Watnick who work in Johns Hopkins University School of Medicine. The complement inhibitor rosmarinic acid (RMA) was purchased from Sigma-Aldrich and suspended in 0.5% sodium carboxymethylcellulose (CMC)/ saline solution for daily gavage. Pkd1 gene knockout mice were induced by oral administration of tamoxifen (150 mg kg 1 day 1) to the nursing mother for three consecutive days at postnatal day (P)10. Both Pkd1 / and Pkd1+/+ mice from the same litters were randomly allocated to four
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groups: Pkd1 / R300, Pkd1 / CON, Pkd1+/+ R300 and Pkd1+/+ CON (six mice in each group). In the two R300 groups, RMA (300 mg kg 1 day 1) was administered to the nursing mother by gavage from P7 to P21. After weaning, from P22 to P35, the same dosage of RMA was administered to the mice directly by gavage. Mice in the CON groups were treated using the same protocol with 0.5% sodium CMC/saline solution instead of RMA. The female to male ratio was 1 : 1 in all groups. A metabolic cage was used to collect a 24-h urine sample at P36. The mice were then killed, a blood sample was collected, and the kidney was harvested; half of the kidney was snap-frozen, and the other half was fixed in 4% paraformaldehyde for morphological examination. Han:SPRD rats Han:SPRD rats were obtained from the Zurich Center for Integrative Human Physiology, University Hospital, Switzerland. Thirty male Han:SPRD heterozygous (Cy/+) rats were randomly assigned (10/group) to two RMA treatment groups [Cy/+ R150 (150 mg kg 1 day 1) and Cy/+ R300 (300 mg kg 1 day 1)] and a control group (Cy/+ CON). Sixteen male Han:SPRD wild-type (+/+) rats were randomly assigned to an RMA treatment group [+/+ R300 (300 mg kg 1 day 1)] and a control group (+/+ CON). From the 4th week after birth, the rats in the RMA treatment groups were treated by gavage with the designated doses of drug for 12 weeks; the rats in the control group received by gavage the same volume of 0.5% sodium CMC/ saline solution. Rats were given unrestricted access to food and water. Blood and urine samples were collected from the RMA- and vehicle-treated rats after 4, 8 and 12 weeks of treatment. Kidneys were harvested (half of each kidney was snapfrozen and the other half was fixed in 4% paraformaldehyde for subsequent morphological analysis) after 12 weeks of treatment. All animal experiments and the collection of human samples were approved by the Ethics Committee of the Second Military Medical University, Shanghai, China. Detection of biochemical indices and renal function The Synchron LX, UniCel DxC 600/800 and Synchron AQUA CAL 1 and 2 Systems were used for quantitative determination of creatinine, blood urea nitrogen (BUN), alanine aminotransferase, aspartate aminotransferase and lipid concentrations in plasma and urine of rats and mice.
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Urinary alternative pathway haemolytic complement activity (AH50) was measured using the rat AH50 enzyme-linked immunosorbent assay kit (Groundwork Biotechnology Diagnostics, Ltd, San Diego, CA, USA) according to the manufacturer’s instructions. Immunohistochemical analysis Complement Paraffin sections (3 lm thick) were cut from renal tissue blocks and incubated with a primary antibody against CFB (1 : 300, R&D systems), C9 (1 : 100, Abcam) or C5b-9 (1 : 200, Sigma-Aldrich) at 4 °C overnight and stained with chromogen 3,3diaminobenzidine (DAB) using the ABC kit (Thermo Scientific, San Jose, CA, USA) according to the manufacturer’s instructions. Ki67 Immunohistochemical staining for Ki67 was performed on 3-lm-thick tissue sections. In brief, the tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed in an autoclave oven. A mouse anti-Ki67 antibody (1 : 600, Abcam) was used as the primary antibody. After applying the primary antibody for 1 h, the sections were washed and then incubated with biotinylated secondary antibody (Vector) for 30 min. This was followed by application of the ABC reagent (Vector). Diaminobenzidine with metal enhancement was used as the detection reagent. For each section, we randomly chose 10 cysts, counted the number of Ki67-positive nuclei and then averaged the number of Ki67-positive nuclei per cyst. Quantification of kidney morphology Six representative images (1009 magnification) stained with haematoxylin and eosin were captured from renal sections of every animal from each group. The images were resized to 800 9 598 pixels, a 5 9 5 grid was placed over each image, and each square counts as one field. The infiltration of inflammatory cells was counted manually and expressed as mean number of cells per 25 fields for each sample. Periodic Acid Schiff (PAS)stained sections were subjected to cyst index analysis, using HistoQuest image analysis software (TissueGnostics, Vienna, Austria), to count the entire cortical region [total area (TA)] and the cyst area (CA) in the renal cortex. The cyst index was calculated as CA/TA 9 100. The interstitial fibrosis index was also measured in the cortical region ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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of PAS-stained sections. The fibrosis index was calculated as the positive pink–purple area/ TA 9 100. All analyses were performed by a na€ıve observer using the HistoQuest software. Statistical analysis Data are presented as means SD. Statistical differences between treatment groups were determined by one-way ANOVA followed by Bonferroni’s test. P < 0.05 was considered to be statistically significant. Results Analysis of urinary glycoproteome in normal control subjects and ADPKD patients: complement components and related proteins We initially hypothesized that complement components in the urinary glycoproteome might serve as important biomarkers for ADPKD. Patients were divided into three groups based on CKD stage: ADPKD stage 1 (GFR ≥ 90 mL min 1 per 1.73 m2), ADPKD stage 2–3 (GFR 30–89 mL min 1 per 1.73 m2) and ADPKD stage 4-5 (GFR ≤ 29 mL min 1 per 1.73 m2); a fourth group of healthy individuals served as the control (Table 1). Ten urine samples from each group were pooled and analysed. We identified a total of 113 different glycoproteins in the urine of ADPKD patients. Details regarding these glycoproteins are provided in the online Supplement (Tables S1–S3 and Figures S1–S3). Of the 113 glycoproteins, there were six complement and complement-related proteins: CFB, SERPING1, C9, C1RL, CD55 and CD59 (Table S4). We then analysed the relative differences in these complement proteins in the urine between controls and ADPKD patients at different CKD stages. For each group, a minimum increase or decrease of 50% (i.e. ≥1.5 or ≤0.67, respectively) compared with controls or between ADPKD stages was considered significant [11]. The levels of CFB, SERPING1 and C9 were increased, whereas the levels of C1RL, CD55 and CD59 were decreased with ADPKD progression (Table 2). Independent validation of changes in urinary complement proteins in ADPKD patients The identification of the aforementioned six complement-related proteins by mass spectroscopy was validation again, because these six proteins were identified as multiple peptides ranging from 12 to 26 peptides for each complement component (Table S4). Nonetheless, it is well known that 474
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changes in glycoprotein levels do not necessarily correlate with the intact proteins that are measured by immunological tests. Thus, we also quantified these six complement-related proteins in the urine using Western blot analysis (Fig. 1) and found that the levels of CFB, SERPING1 and C9 were increased, whereas C1RL, CD55 and CD59 levels were decreased with advancing ADPKD stage. All candidate bands detected by western blotting were then digested and subjected to MS analysis, which confirmed that the bands represented the proteins of interest (Table S5, Figures S2 and S3). Collectively, our data indicate that changes in these six complement proteins are reliably associated with progression of ADPKD. Serum levels of C3 and C4 are not altered in ADPKD patients To determine whether the changes in urinary complement proteins were specifically correlated with the activation of the complement pathway in the kidney or whether they derived from filtered complement components from the blood, we measured the serum levels of C3 and C4, two complement components that are routinely used to monitor systemic complement activity. We found that serum C3 and C4 levels in ADPKD patients were not significantly different from those in normal control subjects (Table 1). These results argue against the possibility of systemic complement activation and suggest that the observed changes in urinary complement components are due to activation of the complement pathway within the kidney. Expression of CFB and C9 in cyst-lining epithelial cells from ADPKD patients and in renal tubular cells from patients with other types of CKD To test the hypothesis that the alternative complement pathway is over-activated in the kidneys of ADPKD patients, we analysed the expression of CFB and C9 in human kidney tissues using immunohistochemistry. Robust CFB signals were detected in the renal tubular and cyst-lining epithelial cells from ADPKD patients, and a few positive signals were observed in renal tubular cells from patients with diabetic nephropathy (DN) and IgAN, whereas no positive signal was detected in the renal tubular of patients with minimal change disease (MCD) or LN, or in normal control subjects. C9 signals were significantly increased in the cyst-lining epithelial cells from ADPKD patients, with minor increases in tubular cells from IgAN and DN patients, but no expression in patients with MCD or LN or in control subjects (Fig. 2a).
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Table 2 Complement-related protein expression in urine samples from ADPKD patients and control subjects as analysed by liquid chromatography–mass spectrometry ADPKD
ADPKD
ADPKD
Accession
stage 1 vs.
stages 2–3
stages 4–5
Names
number
control
vs. control
vs. control
CFB Complement factor B
IPI00639937.1
1.1
1.56
4.71
5.17
CFB Isoform 1 of Complement
IPI00019591.1
factor B precursor Fragment C9 Complement component C9 precursor
IPI00022395.1
1.17
2.77
SERPING1 Complement component 1 inhibitor precursor
IPI00879931.1
1.23
1.49
1.54
C1RL 48 kDa protein
IPI00872573.1
1.4
0.94
0.63
0.65
0.70
0.57
1.06
0.98
0.61
C1RL cDNA FLJ14022 fis, clone HEMBA1003538
IPI00795055.1
weakly similar to COMPLEMENT C1R COMPONENT C1RL Complement C1r subcomponent-like protein precursor
IPI00009793.4
CD55 Decay-accelerating factor splicing variant 5
IPI00382926.2
CD55 Decay-accelerating factor splicing variant 1
IPI00784169.1
CD59 glycoprotein precursor
IPI00011302.1
Data are presented as ratios, which are the stages of autosomal dominant polycystic kidney disease (ADPKD) group divided by the control group respectively. Ratios of ≥1.5 and ≤0.67 denote significant differences from control. IPI, international protein index.
Western blot analysis results further confirmed the upregulation of CFB and C9 in the kidney tissues from ADPKD patients (Fig. 2b). These results demonstrate that the alternative pathway is clearly overactivated in renal cyst tissues of ADPKD patients. Elevated levels of C9 in cyst-lining epithelial cells from conditional Pkd1 / knockout mice To probe whether the complement system is actively involved in the initiation of ADPKD or contributes to its progression, an animal model is required. As an initial step, we measured C9 expression in an Mx1Cre+Pkd1null/flox mouse model [12]. After administration of the interferon inducer polyinosinicpolycytidylic acid (62.5 lg) for five consecutive days at P7 by intraperitoneal injection, kidneys were harvested at 11 weeks of age. No significant difference in C9 expression between normal and precystic kidney tissues was found with immunofluorescence staining, whereas C9 immunoreactivity was highly positive in the cyst-lining epithelial cells of cystic kidney tissues (Fig. 2c). Pharmacological inhibition of the complement pathway attenuates disease progression in conditional Pkd1 / knockout mice We then examined the effect of the complement system inhibitor RMA on disease progression in
conditional Pkd1 / mice. After 4 weeks (P7 to P35) of RMA (300 mg kg 1 day 1) treatment (Fig. 3a), BUN was significantly decreased in RMA-treated Pkd1 / mice (9.6 1.3 mmol L 1) compared with vehicle-treated Pkd1 / animals (43.2 7.8 mmol L 1); the same effect was observed for plasma creatinine levels (17.4 1.1 lmol L 1 vs. 35.8 9.1 lmol L 1; Fig. 3b). In RMA-treated Pkd1 / mice, the cyst index was reduced by 59.8% and the two kidneys/body weight ratio (2K/ BW%) was reduced by 60.3%, compared with vehicle-treated Pkd1 / mice, without any body weight loss (Fig. 3b and c). Complement factor B and C5b-9 expressions in renal tissues from Pkd1 / mice were upregulated compared with Pkd1+/+ mice. RMA treatment reduced CFB and C5b-9 expression in Pkd1 / mice compared with vehicle-treated Pkd1 / animals (Fig. 3d). Pharmacological inhibition of the complement pathway attenuates disease progression in Han:SPRD rats Compared with the vehicle-treated control animals, BUN and plasma creatinine levels were significantly decreased in rats treated with different dosages of RMA (150 or 300 mg kg 1 /day 1; Fig. 4a), indicating that RMA treatment improved kidney function in Han:SPRD Cy/+ rats. The 2K/BW ratio and cystic index were significantly improved in ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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Control
(a)
ADPKD stage 2~3
ADPKD stage 1
ADPKD stage 4~5
CFB
C9
SERPING1
C1RL
CD55
CD59
CFB
(b)
C9
3500
*
3000 2500 2000
*
2500 2000 1500
1500 1000 500 0
1000 500 0
Control
ADPKD stage 1
ADPKD ADPKD stage 2–3 stage 4–5
Control
3000
*
2500 2000 1500 1000 500
Control
ADPKD stage 1
ADPKD ADPKD stage 2–3 stage 4–5
C1RL
SERPING1
0
ADPKD stage 1
ADPKD ADPKD stage 2–3 stage 4–5
1800 1600 1400 1200 1000 800 600 400 200 0
* Control
CD55
ADPKD stage 1
ADPKD ADPKD stage 2–3 stage 4–5
CD59 2500
1400 1200 1000 800 600 400
2000 1500
*
200 0
Control
ADPKD stage 1
*
ADPKD ADPKD stage 2–3 stage 4–5
*
1000 500 0
Control
ADPKD stage 1
ADPKD ADPKD stage 2–3 stage 4–5
Fig. 1 Western blotting (a) and densitometric (b) analysis of complement-related protein expression in urine samples from autosomal dominant polycystic kidney disease (ADPKD) patients and control subjects. The protein expression levels of complement factor B (CFB), C9 and SERPING1 were increased, whereas those of C1RL, CD55 and CD59 were decreased with ADPKD progression. 476
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(a)
(b)
(c)
Fig. 2 Immunohistochemical staining (a) and Western blotting and densitometric analysis (b) of complement factor B (CFB) and C9 in the kidney of autosomal dominant polycystic kidney disease (ADPKD) patients and immunofluorescence staining of C9 in normal, precystic and cystic kidney tissues from conditional Pkd1 / knockout mice (c). No positive signal for either CFB or C9 was detected in the renal cortex of normal control subjects or of MCN and LN patients, whereas slight CFB and C9 signals were detected in DN and IgAN patients, with robust signals in the renal tubular and cyst epithelial cells of ADPKD patients (a). Compared with normal control subjects, the protein expression levels of both CFB and C9 in the kidney tissues from ADPKD patients were significantly increased (*P < 0.05 vs. normal control subjects; b). There was a significant upregulation of C9 in cystic kidney compared to normal kidney; C9 was mainly localized to cyst-lining epithelial cells (c). MCD, minimal change disease, LN, lupus nephritis; DN, diabetic nephropathy; IgAN, IgA nephropathy.
RMA-treated Han:SPRD Cy/+ rats with either dose (P < 0.05) compared with the control group after 12 weeks of treatment (Fig. 4b,c). Using immunohistochemistry, we also found robust positive signals for CFB and C5b-9 in the renal tubular and cyst-lining epithelial cells in vehicle-treated Cy/+ rats, which were significantly inhibited by RMA
treatment; these complement proteins were not detectable in Han:SPRD +/+ rats (Fig. 4d). To confirm the efficacy of RMA in inhibiting complement activation, we measured the haemolytic activity (AH50) of the complement pathway in rat urine after 12 weeks of treatment. We found that ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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(a)
(b)
Fig. 3 Therapeutic effect of rosmarinic acid (RMA) in conditional Pkd1 / knockout mice. Schematic diagram of the experimental design (a); blood urea nitrogen (BUN), serum creatinine (sCr), body weight, total kidney weight, two kidneys/ body weight ratio (2K/BW%) and cyst index after 4 weeks of RMA treatment (b); Periodic Acid Schiff (PAS) staining of kidney sections (c); renal expression of complement factor B (CFB) and C5b-9 by immunohistochemical staining (d). RMA treatment significantly reduced BUN, sCr, total kidney weight, 2K/BW% and cyst index without affecting body weight. Expression of CFB and C5b-9 was inhibited after RMA treatment.
the AH50 activity in the urine was significantly lower in Han:SPRD Cy/+ rats treated with RMA than in vehicle-treated animals (Table S6). In addition, there were no significant differences in liver function or triglyceride and cholesterol levels between the control and RMA-treated groups (Table S6), indicating that RMA treatment did not have any severe side effects on either liver function or lipid metabolism. Antiproliferation, inhibition of inflammatory cell infiltration and reduced fibrosis by inhibition of the complement system in conditional Pkd1 / mice and Han:SPRD rats To explore how inhibition of the complement system may retard cystic disease progression, we determined the effect of RMA on tubular epithelial cell proliferation, tubular interstitial inflammatory cell infiltration and fibrosis. The number of Ki-67478
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positive nuclei was significantly increased in the cyst-lining epithelial cells in kidney tissues from Pkd1 / mice and Han:SPRD Cy/+ rats compared with tubular epithelial cells in kidney tissues from Pkd1+/+ mice and Han:SPRD +/+ rats. RMA treatment led to a significant reduction in the number of Ki-67-positive nuclei in cyst-lining epithelial cells in Pkd1 / mice and Han:SPRD Cy/+ rats compared with vehicle-treated animals (Fig. 5a and Table 3), indicating that inhibition of the complement pathway is linked to a decrease in the proliferation of cyst-lining epithelial cells. Inflammatory cell infiltration in the renal interstitial area was greatly increased in kidneys from Pkd1 / mice and Han:SPRD Cy/+ rats compared with those from Pkd1+/+ mice and Han:SPRD +/+ rats and was significantly decreased after RMA treatment compared with vehicle treatment in
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(c)
(d)
Fig. 3 Continued.
Pkd1 / mice and Han:SPRD Cy/+ rats (Fig. 5b and Table 3); thus, inhibition of the complement pathway with RMA ameliorated the inflammatory cell infiltration in tubular interstitium. Renal fibrosis was assessed by PAS staining. There was a much larger amount of PAS-positive staining in the interstitial area of kidney tissues from
Pkd1 / mice and Han:SPRD Cy/+ rats compared with Pkd1+/+ mice and Han:SPRD +/+ rats. Similarly, the area between cysts and tubules was much larger in Pkd1 / mice and Han:SPRD Cy/+ rats than in Pkd1+/+ mice and Han:SPRD +/+ rats. After RMA treatment, the amount of positive PAS staining was decreased and the area between cysts and tubules became smaller in Pkd1 / mice and ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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(a)
(b)
Fig. 4 Therapeutic effect of rosmarinic acid (RMA) in Han:SPRD rats. Schematic diagram of the experimental design (a); blood urea nitrogen (BUN), serum creatinine (sCr), body weight, total kidney weight, two kidneys/body weight ratio (2K/BW %) and cyst index after 12 weeks of RMA treatment (b); Masson staining of kidney sections (c); renal expression of complement factor B (CFB), C9 and C5b-9 by immunohistochemical staining (d). RMA treatment significantly reduced BUN, sCr, total kidney weight, 2K/BW% and cyst index without affecting body weight. CFB, C9 and C5b-9 expression were inhibited after RMA treatment. Magnification: 4009 in C; scale bar: 100 lm in D. CMC-Na, sodium carboxymethylcellulose; R150, 150 mg kg 1 day 1 RMA; R300, 300 mg kg 1 day 1 RMA; CON, control.
Han:SPRD Cy/+ rats, demonstrating that inhibition of the complement system with RMA was associated with a decrease in fibrosis (Fig. 5c and Table 3). Discussion The results of the present study show that excessive activation of the alternative complement pathway is associated with ADPKD progression and that treatment with the complement inhibitor RMA had a beneficial effect on disease progression in experimental models of polycystic kidney disease. Previous studies have already demonstrated that 480
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mRNA transcripts for components of the complement system are over-expressed in kidney tissues from Han:SPRD rats [5]. Furthermore, it has been shown that substantial amounts of complement proteins are present in renal cyst fluid of ADPKD patients [6]. These preliminary findings have prompted the following questions: (i) Does complement activation occur within the kidney parenchyma of ADPKD patients? (ii) Which specific pathways of the complement system are involved? (iii) Is activation of the complement system specific for ADPKD? (iv) Is the over-expression of complement components an initial event or a sustained influence on ADPKD progression? and
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(c)
(d)
Fig. 4 Continued.
(v) finally, what are the mechanisms by which over-activation of the complement system causes ADPKD progression? To exclude the possibility that the changes in urinary complement components are not the result
of systemic complement activation, we measured serum C3 and C4 levels in ADPKD patients and normal control subjects. As serum C3 and C4 levels were normal, it appears that the changes in complement components in ADPKD occur within the kidney. Furthermore, the 24-h urinary protein ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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Table 3 Effect of rosmarinic acid treatment on Ki-67 immunohistochemistry staining, inflammatory cell infiltration and fibrosis in kidney tissues from conditional Pkd1 / knockout mice and Han:SRPD rat Ki-67 positive nuclear
Interstitial inflammatory cells
Fibrosis index (%)
Conditional Pkd1 knock-out mice Pkd1
/
R300
1.1 0.1***
Pkd1
/
CON
5.9 0.3
Pkd1+/+ R300 +/+
Pkd1
CON
6.4 0.3** 12.5 1.4
3.9 1.4** 12.3 1.7
0.5 0.1***
1.1 0.2***
1.6 0.9***
0.5 0.1***
1.2 0.2***
3.8 1.0***
Han:SPRD rat Cy/+ R300
1.7 0.5^^
23.8 1.8^^
Cy/+ CON
2.6 0.7
42.2 1.6
+/+ R300
1.0 0.1
4.6 0.1
6.3 1.3^^^
+/+ CON
0.9 0.0
4.3 0.1
7.5 2.1^^^
^^^
**P < 0.01, ***P < 0.001 vs. Pkd1
^^^
/
Using quantitative urinary glycoproteomics and various confirmatory techniques, we have shown for the first time that the levels of the specific complement components CFB, SERPING1 and C9 are increased, whereas C1RL, CD55 and CD59 levels are decreased in urine samples from ADPKD patients. SERPING1 is one of the key regulators of the complement pathways, strongly inhibiting C1r and C1s in the classical pathway as well as MASP-1 and MASP-2 in the MBL pathway [13]. Thus, SERPING1 not only controls the initiation of the classical pathway but also inhibits the early activation steps of the MBL pathway. The increase in SERPING1 found in the present study suggests that these two pathways are unlikely to be activated in ADPKD patients. Complement factor B is a crucial factor for initiating and sustaining the activation of the alternative pathway [14]. CD55 is an inhibitor of C3 convertase, C9 participates in the formation of C5b-9, and CD59 inhibits the function of C9. The decreases in CD55 and CD59 are conducive to the activation of the complement pathway (Fig. 6). Therefore, the observed alterations in the levels of these six complement-related proteins in the urine of ADPKD patients are consistent with the selective activation of the alternative complement pathway. ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
^^^
17.7 5.3
CON group; ^^P < 0.01, ^^^P < 0.001 vs Han:SPRD Cy/+ CON group.
excretion was within normal limits in all groups of ADPKD patients (Table 1), suggesting again that the complement proteins present in the urine of ADPKD patients are not the result of leakage through the glomerular barrier, but rather originate from renal tubular cells and cyst-lining epithelial cells.
482
^^^
7.9 3.9^^
Complement factor B is a crucial factor for initiating and sustaining the activation of the alternative complement pathway, and C9 is important for the formation of the final membrane attacking complex C5b-9; therefore, next we determined the levels of CFB and C9 in kidney tissues of patients with ADPKD and compared them to levels in patients with other types of chronic kidney disease (MCD, DN, IgAN and LN). Immunohistochemical staining revealed that CFB and C9 were markedly overexpressed in tubular and cyst-lining epithelial cells in kidney tissues from ADPKD patients, but only slightly increased in renal tubular cells of kidney biopsy samples from DN and IgAN patients. In addition, no staining was observed in patients with MCD and LN. These findings suggest that the overexpression of CFB and C9 is specific to patients with ADPKD. We also found that the increases in urinary complement components CFB, SERPING1 and C9 and the decreases in C1RL, CD55 and CD59 were correlated with the different stages of ADPKD. C9 is not expressed in normal and precystic kidneys, but is highly expressed in the cyst-lining epithelial cells of cystic kidney tissues. We therefore investigated the effect of RMA which has been reported to inhibit complement activation predominately by reacting covalently with the activated thioester in nascent C3b, thus blocking C3b attachment to complement-activating surfaces. We found that RMA inhibited the expression of CFB and C9, reduced cyst index and 2K/BW% and improved renal function in conditional Pkd1 / knockout mice and Han:SPRD Cy/+ rats. These data strongly
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(a)
(b)
Fig. 5 Mechanism of complement-mediated progression of autosomal dominant polycystic kidney disease (ADPKD). Ki-67 immunohistochemical staining (a). The Ki-67-positive signal was black in Pkd1 mice and dark brown in Han:SRPD rats due to the different visualization reagents. Inflammatory cell infiltration determined in haematoxylin-and-eosin-stained sections (b) and fibrosis visualised by PAS staining (c). The PAS-positive signal was pink– purple. R300, 300 mg kg 1 day 1 RMA, rosmarinic acid; CON, control.
(c)
suggest that over-activation of the alternative complement pathway is important in the progression of ADPKD, rather than an as an inducer of cystogenesis. The mechanisms that lead to over-activation of the alternative complement pathway in ADPKD are complex. The number of Ki-67-positive cells and interstitial inflammatory cell infiltration and the
degree of interstitial fibrosis were all reduced by RMA treatment in kidney tissues from conditional Pkd1 / knockout mice and Han:SPRD Cy/+ rats, indicating that ADPKD progression which is mediated by the over-activation of the alternative complement pathway is due at least in part to proliferation of cyst-lining epithelial cells, interstitial inflammatory cell infiltration and interstitial fibrosis. ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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urinary glycoproteins and achieved an enrichment of these low-abundance proteins with the use of hydrazide beads. This approach allowed enrichment of glycoproteins and reduction in the overall range of proteins. Amongst 139 identified proteins, a total of 113 were genuine glycoproteins, which reflects a specificity of 81.3%. To the best of our knowledge, our study is the first comprehensive and systematic characterization of the urinary glycoproteome (Table S1). In a few previous studies, the levels of total, membrane and phosphorylated proteins have been analysed in urine with or without fractionation [17, 18]. Such unbiased profiling tends to preferentially identify more abundant proteins. In our study, 21 of the 261 glycopeptides have not been annotated by the current database (Table S3).
Fig. 6 Schematic diagram of excessive activation of the alternative complement pathway in autosomal dominant polycystic kidney disease (ADPKD) progression. Red denotes upregulation; green denotes downregulation; ? denotes stimulation; denotes inhibition; pink rectangle denotes the effect of rosmarinic acid (RMA). CFB, complement factor B.
A limitation of this study is that RMA is not a specific complement inhibitor, as it also inhibits glutathione reductase, glucose-6-phosphate dehydrogenase and the caspase-1 pathway. A more specific inhibitor of the alternative complement pathway or the cross-breeding between CFB knockout mice and Pkd1 knockout mice would be required to confirm the unique role of the alternative complement pathway in ADPKD progression. Nevertheless, our findings provide hope that inhibitors of the same type as RMA could be useful agents to retard disease progression in patients with ADPKD at least in part by blocking the alternative complement system. It is worth mentioning a major technical obstacle to the identification of proteins by proteomic methodologies in body fluids with a vast array of proteins (including plasma and urine): mass spectrometry is typically biased towards abundant proteins [15]. Several approaches have been employed to overcome this problem [16]. In the present study, we focused on the subproteome of 484
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In conclusion, using a robust quantitative proteomics screen, we identified 113 different glycoproteins and 261 glycopeptides in the urine; moreover, we found significant expression changes in six complement components in the urine from ADPKD patients. Excessive activation of the alternative complement pathway was observed in cystic kidneys of ADPKD patients, conditional Pkd1 / mice and Han:SPRD rats and may exacerbate ADPKD progression. The complement inhibitor RMA effectively ameliorated the pathological changes and delayed the deterioration of renal function in conditional Pkd1 / mice and Han:SPRD rats, which suggests that targeting the complement system may be a novel therapeutic strategy for ADPKD. Conflict of interest statement The authors declare that they have no competing interests. Acknowledgements This work was supported by the National High Technology Research and Development Program 863 Project (2007AA02Z3Z1), National Natural Science Foundation of China General Projects (30971368, 30871179), Shanghai Key Discipline Project (B902), Major National Science and Technology Project (2009ZX09102), Shanghai Science and Technology Commission Major Research Project (11431920800), Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents, Qianjiang Talents Project of Science and Technology Department in Zhejiang Province, Shanghai International Science and Technology
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Cooperation Fund Project (0954070200) to CM; the Shanghai Science and Technology Committee General Project (09ZR1410400) to XW; National Institute of Child Health and Human Development (P30 HD02274) to RPB; and the Swiss National Science Foundation (320030-144093 to RPW and 310030132597 to ALS).
References 1 The International Polycystic Kidney Disease Consortium. Polycystic kidney disease: the complete structure of the PKD1 gene and its protein. The International Polycystic Kidney Disease Consortium. Cell 1995; 81: 289–98. 2 Mochizuki T, Wu G, Hayashi T et al. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science 1996; 272: 1339–42. 3 Patel V, Chowdhury R, Igarashi P. Advances in the pathogenesis and treatment of polycystic kidney disease. Curr Opin Nephrol Hypertens 2009; 18: 99–106. 4 Wuthrich RP, Mei C. Aquaretic treatment in polycystic kidney disease. N Engl J Med 2012; 367: 2440–2. 5 Burtey SJ, Riera M, Fontes M. Overexpression of complement-component genes in Han:SPRD rats a model of polycystic kidney disease. Kidney Int 2008; 73: 1324–5; author reply 5. 6 Lai X, Bacallao RL, Blazer-Yost BL, Hong D, Mason SB, Witzmann FA. Characterization of the renal cyst fluid proteome in autosomal dominant polycystic kidney disease (ADPKD) patients. Proteomics Clin Appl 2008; 2: 1140–52. 7 Pei Y, Obaji J, Dupuis A et al. Unified criteria for ultrasonographic diagnosis of ADPKD. J Am Soc Nephrol 2009; 20: 205–12. 8 Shi M, Bradner J, Bammler TK et al. Identification of glutathione S-transferase pi as a protein involved in Parkinson disease progression. Am J Pathol 2009; 175: 54–65. 9 Hong Z, Shi M, Chung KA et al. DJ-1 and alpha-synuclein in human cerebrospinal fluid as biomarkers of Parkinson’s disease. Brain 2010; 133: 713–26. 10 Piontek KB, Huso DL, Grinberg A et al. A functional floxed allele of Pkd1 that can be conditionally inactivated in vivo. J Am Soc Nephrol 2004; 15: 3035–43. 11 Zhang J, Goodlett DR, Peskind ER et al. Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid. Neurobiol Aging 2005; 26: 207–27. 12 Takakura A, Contrino L, Beck AW, Zhou J. Pkd1 inactivation induced in adulthood produces focal cystic disease. J Am Soc Nephrol 2008; 19: 2351–63. 13 Cugno M, Zanichelli A, Foieni F, Caccia S, Cicardi M. C1-inhibitor deficiency and angioedema: molecular mechanisms and clinical progress. Trends Mol Med 2009; 15: 69–78. 14 Le Quintrec M, Lionet A, Kamar N et al. Complement mutation-associated de novo thrombotic microangiopathy following kidney transplantation. Am J Transplant 2008; 8: 1694–701.
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15 Wang L, Li F, Sun W et al. Concanavalin A-captured glycoproteins in healthy human urine. Mol Cell Proteomics 2006; 5: 560–2. 16 Ghosh D, Krokhin O, Antonovici M et al. Lectin affinity as an approach to the proteomic analysis of membrane glycoproteins. J Proteome Res 2004; 3: 841–50. 17 Li QR, Fan KX, Li RX et al. A comprehensive and non-prefractionation on the protein level approach for the human urinary proteome: touching phosphorylation in urine. Rapid Commun Mass Spectrom 2010; 24: 823–32. 18 Lai ZW, Steer DL, Smith AI. Membrane proteomics: the development of diagnostics based on protein shedding. Curr Opin Mol Ther 2009; 11: 623–31. Correspondence: Prof. Changlin Mei, Kidney Institute, Department of Nephrology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, China. (Fax: (0086)-21-63521416; e-mail: [email protected]). Prof. Rudolf P. W€ uthrich, Division of Nephrology, University Hospital Zurich, University of Zurich, 8091 Zurich, Switzerland. (Fax: (0041)-44-2554593; e-mail: [email protected])
Supporting Information Additional Supporting Information may be found in the online version of this article: FigureS1. Gene ontology analysis of glycoprotein identified in human ADPKD urine. FigureS2. Characterization of CFBin human urine. Figure S3. Characterization of SERPING1, C9, C1RL, CD55 and CD59in human urine. Table S1. Glycoproteins and Glycopeptides Identified in Human Urine. Table S2. Glucoproteins identified in human urine of previous investigations. Table S3. Glycopeptides unannotated by current database. Table S4. The 6 complement-related proteins identified by multiple peptides. Table S5. Human CFB,SERPING1,C9,C1RL,CD55 and CD59peptides identified by mass spectrometry in urine.
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Excessive activation of the alternative complement pathway in autosomal dominant polycystic kidney disease Z. Su1,*,†, X. Wang1,*, X. Gao1,*, Y. Liu2, C. Pan3, H. Hu1, R. P. Beyer4, M. Shi3, J. Zhou5, J. Zhang3, A. L. Serra2,6, R. P. W€uthrich2,6 & C. Mei1 From the 1Kidney Institute, Department of Nephrology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China; 2Institute of Physiology, University of Zurich, Zurich, Switzerland; 3Division of Neuropathology, Harborview Medical Center, University of Washington School of Medicine; 4Department of Environmental & Occupational Health Sciences, University of Washington, Seattle, WA; 5 Harvard Institutes of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; 6Division of Nephrology, University Hospital Zurich, Zurich, Switzerland †Present address: Division of Nephrology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, Zhejiang, China
Abstract. Su Z, Wang X, Gao X, Liu Y, Pan C, Hu H, Beyer RP, Shi M, Zhou J, Zhang J, Serra AL, W€ uthrich RP, Mei C (Second Military Medical University, Shanghai; University of Zurich, Zurich; University of Washington School of Medicine, Seattle, WA; University of Washington, Seattle, WA; Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; University Hospital Zurich, Zurich). Excessive activation of the alternative complement pathway in autosomal dominant polycystic kidney disease. J Intern Med 2014; 276: 470–485. Objectives. The complement system is involved in many immune complex-mediated kidney diseases, yet its role in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) has not been examined in detail. Methods and Results. Screening of the glycoproteome of urine samples from ADPKD patients revealed that levels of complement factor B (CFB), serpin peptidase inhibitor, complement component 1 inhibitor (SERPING1) and complement component 9 (C9) increased, whereas complement component 1, r subcomponent-like (C1RL), CD55 and CD59 levels decreased with disease progression. Immunostaining and Western blot analysis confirmed the enhanced expression of CFB and C9 in cystic
Introduction Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development and growth of innumerable cysts that originate from the tubular epithelium of nephrons [1]. Mutations in the PKD1 or PKD2 genes are the primary cause of ADPKD [1, 2], resulting in disturbances of multiple *These authors contributed equally to this paper.
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kidneys from ADPKD patients. Immunostaining also showed that the expressions of CFB and C9 in renal biopsy tissues from patients with other types of chronic kidney disease were lower than in tissues from ADPKD patients. The effect of the complement inhibitor rosmarinic acid (RMA) was evaluated in Pkd1 / mice and Han:SPRD Cy/+ rats. Compared with vehicle-treated Pkd1 / animals, RMA-treated mice had significantly lower serum creatinine ( 50%) and blood urea nitrogen ( 78%) levels, two kidneys/body weight ratio ( 60%) and renal cystic index ( 60%). Similar results were found in Cy/+ rats. Lower numbers of Ki67-positive nuclei and inflammatory cells and reduced fibrosis were observed in both animal models upon treatment with RMA. Conclusions. These results suggest that excessive activation of the alternative complement pathway is associated with ADPKD progression, probably mediated by cyst-lining epithelial cell proliferation, tubulointerstitial inflammatory cell infiltration and fibrosis. Targeting the complement system might represent a new therapeutic strategy for ADPKD. Keywords: autosomal dominant polycystic kidney disease, complement, complement inhibitor, glycoproteomics, urine.
cellular signalling pathways. The pathophysiological changes in the disease consist of cell proliferation and apoptosis, fluid secretion, altered cellular polarity and interstitial fibrosis, leading to renal failure in 50% of patients by the age of 60 [3, 4]. The complement system is involved in many types of immune complex-mediated kidney disease, such as IgA nephropathy (IgAN), lupus nephritis (LN)
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and membranous and membranoproliferative nephropathy, yet its role in the pathogenesis of ADPKD is unknown. The complement system can be activated via three different pathways: the classical, alternative and mannose-binding lectin (MBL) pathways. Although essential to defend against pathogens, excessive activation of the complement system causes inflammation and tissue injury. Recently, using microarray analysis, Burtey et al. [5] found that nine different complement component genes were over-expressed in the kidneys of Han:SPRD rats, which is a model for inherited polycystic kidney disease. Furthermore, Lai et al. [6] identified seven complement proteins in the cystic fluid from patients with ADPKD. The purpose of the present study was to examine the activity of the complement system as a function of ADPKD progression and to explore the possibility that this system could provide a novel therapeutic target for ADPKD. Methods Study participants Ten healthy adults served as control subjects. Thirty patients with ADPKD were recruited from Shanghai Changzheng Hospital. The diagnostic criteria for ADPKD included a positive family history of ADPKD and characteristic findings on B-ultrasonography [7] as well as the absence of primary disease of another system; in addition, denaturing high-performance liquid chromatography was used to reveal a PKD1 mutation. Based on the chronic kidney disease (CKD) stages, patients were classified as ADPKD stage 1, ADPKD stage 2–3 and ADPKD stage 4–5 (with 10 patients per group). These four groups (including the control group) were comparable with regard to age and gender (Table 1). Informed consent was obtained from all participants. Human urine collection For collection of the second-morning urine samples, subjects in the four groups fasted (food and fluid) from 22:00 on the previous day until the second void on the study morning. Fresh mid-stream urine was centrifuged, and the supernatant was collected and immediately stored at 80 °C. Protease inhibitors were not used in the urine collection.
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and centrifuged at 4 °C. The supernatant was collected and stored at 80 °C. Analysis of the urinary glycoproteome Total protein was extracted using acetone precipitation from 10 mL urine from each subject. An equal amount of protein from the 10 individual samples in each group was combined, and 120 lg protein from each group was digested with trypsin in parallel and then labelled with isobaric tag for relative and absolute quantification (iTRAQ) reagents (Applied Biosystems, Foster City, CA, USA) as described previously [8]. The first experiment used 4-Plex iTRAQ, with reagents 114, 115, 116 and 117 added to the control, ADPKD stage 1, ADPKD stage 2–3 and ADPKD stage 4–5 groups, respectively. To reduce experimental and intergroup deviations, a second experiment was performed using 8-Plex iTRAQ, with reagents 113, 114, 115 and 116 added to the control, ADPKD stage 1, ADPKD stage 2–3 and ADPKD stage 4–5 groups, respectively, as a repeat experiment reagents 117, 118, 119 and 121 were added to the control, ADPKD stage 1, ADPKD stage 2–3 and ADPKD stage 4–5 respective groups, which as the second repeat experiment. The iTRAQlabelled digests were combined, loaded onto a C18 column (Waters, Milford, MA, USA) to desalt, and were then oxidized with 10 mmol L 1 sodium periodate at room temperature for 1 h. After a second desalting step in a C18 column, the samples were incubated with affinity gel hydrochlorothiazide resin (Bio-Rad Laboratories, Berkeley, CA, USA) at room temperature for 24 h, washed and reacted with peptide-N-glycosidase F (PNGase F; New England BioLabs, Beverly, MA, USA) at 37 °C for 48 h. The supernatant was collected, and the specimens were dissolved in 0.5% trifluoroacetic acid (TFA) and separated with reverse-phase chromatography. Mass spectrometry (MS) was performed with a 4800-matrix-assisted laser desorption ionization time of flight tandem mass spectrometer (MALDITOF/TOF; Applied Biosystems). The MS spectra were searched against the international protein index human protein database (version 3.18) using the Proteinpilot 2.0 software (Applied Biosystems) for peptide and protein identification and quantification. Western blot analysis
Human blood sample collection For the serum C3 and C4 assays, fasting venous blood (5 mL) was collected in the early morning
Urine concentrations of complement factor B (CFB), complement component 1, r subcomponent-like (C1RL), serpin peptidase inhibitor, complement ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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Table 1 Clinical characteristics of ADPKD patients and control subjects
Sex M/F) Age (years) 24-h urine protein (mg) SCr (lmol L
1
)
Control
ADPKD stage 1
ADPKD stage 2–3
ADPKD stage 4–5
6/4
6/4
6/4
6/4
48 3
44 5
60 14
67 16
79 26
91 31
62 11
66 16
160 20*
445 30*
58 15*
16 8*
1
)
1.1 0.4
1.1 0.2
1.2 0.2
1.0 0.3
Serum C4(g L
1
)
0.2 0.1
0.2 0.1
0.3 0.1
0.3 0.1
1
per 1.73 m2)
123 17
50 4
Serum C3(g L
eGFR (mL min
130 6
46 5
The data shown are the mean SD. ADPKD, autosomal dominant polycystic kidney disease; SCr, serum creatinine; eGFR, estimated glomerular filtration rate using modification of diet in renal disease (MDRD) formula. *P < 0.05 compared with control. There were no significant differences in serum C3 and C4 levels between the four groups.
component 1 inhibitor (SERPING1), CD55, CD59 and C9 were measured by Western blot analysis. In brief, an equal amount of total protein from each group was collected, and the glycoproteins were enriched as described above. Glycoproteins (20 lg per group) were separated by 8–16% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride membranes. The membranes were blocked and probed overnight at 4 °C with 1 : 1000 goat anti-human CFB (R&D systems, Minneapolis, MN, USA), 1 : 350 rabbit anti-human C1RL (SigmaAldrich, St. Louis, MO, USA), 1 : 200 mouse antihuman SERPING1 (Abnova Corporation), 1 : 1000 mouse anti-human C9 (Abcam, Cambridge, UK), 1 : 200 mouse anti-human CD55 (Abcam) or 1 : 500 mouse anti-human CD59 (Novus Biologicals) antibodies, followed by peroxidase-conjugated anti-goat, anti-mouse or anti-rabbit IgG (1 : 2000) at room temperature for 1 h. An enhanced chemiluminescence (Amersham Pharmacia) was used for protein band visualization. CFB and C9 in the human and rat kidney tissues were also detected by western blotting as described above, using 1 : 1000 goat anti-human CFB or 1 : 1000 mouse antihuman C9 antibodies. Normal human renal tissues were obtained from the uninvolved pole of kidneys that were removed due to a localized cancer. Autosomal dominant polycystic kidney disease renal tissues were obtained from patients who required nephrectomy of one or both cystic kidney (s) to better accommodate a renal allograft. In-gel digestion and MS validation Enriched urinary glycoproteins from each group were separated by SDS-PAGE, fixed and stained with Coomassie blue. The protein bands to be 472
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identified (CFB, C1RL, SERPING1, CD55, CD59 and C9) were excised from the gel, cut into 1-mm3 pieces and incubated with 50 lL bleaching solution (50% acetonitrile and 25 mmol L 1 bicarbonate) at room temperature for 30 min. After removing the bleaching solution, the above procedures were repeated until the gel became transparent. The gel was then dried in a vacuum centrifuge to obtain white granules, to which 1 mL trypsin (20 ng lL 1 dissolved in 20 mmol L 1 bicarbonate) was added for overnight digestion at 30 °C. The peptide was obtained after washing and desalting and then dried and re-dissolved in 2 lL 0.2% TFA solution for MS identification (MALDI-TOF/TOF) as previously described [9]. Immunoturbidity of serum C3 and C4 Serum C3 and C4 levels in ADPKD patients were detected by immunoturbidity using C3 and C4 assay kits, respectively, according to the manufacturer’s instructions (Dade Behring, Newark, NJ, USA). Conditional Pkd1
/
mice
Conditional Pkd1 / mice [10] were a gift from Professor G. Germino, and T. Watnick who work in Johns Hopkins University School of Medicine. The complement inhibitor rosmarinic acid (RMA) was purchased from Sigma-Aldrich and suspended in 0.5% sodium carboxymethylcellulose (CMC)/ saline solution for daily gavage. Pkd1 gene knockout mice were induced by oral administration of tamoxifen (150 mg kg 1 day 1) to the nursing mother for three consecutive days at postnatal day (P)10. Both Pkd1 / and Pkd1+/+ mice from the same litters were randomly allocated to four
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groups: Pkd1 / R300, Pkd1 / CON, Pkd1+/+ R300 and Pkd1+/+ CON (six mice in each group). In the two R300 groups, RMA (300 mg kg 1 day 1) was administered to the nursing mother by gavage from P7 to P21. After weaning, from P22 to P35, the same dosage of RMA was administered to the mice directly by gavage. Mice in the CON groups were treated using the same protocol with 0.5% sodium CMC/saline solution instead of RMA. The female to male ratio was 1 : 1 in all groups. A metabolic cage was used to collect a 24-h urine sample at P36. The mice were then killed, a blood sample was collected, and the kidney was harvested; half of the kidney was snap-frozen, and the other half was fixed in 4% paraformaldehyde for morphological examination. Han:SPRD rats Han:SPRD rats were obtained from the Zurich Center for Integrative Human Physiology, University Hospital, Switzerland. Thirty male Han:SPRD heterozygous (Cy/+) rats were randomly assigned (10/group) to two RMA treatment groups [Cy/+ R150 (150 mg kg 1 day 1) and Cy/+ R300 (300 mg kg 1 day 1)] and a control group (Cy/+ CON). Sixteen male Han:SPRD wild-type (+/+) rats were randomly assigned to an RMA treatment group [+/+ R300 (300 mg kg 1 day 1)] and a control group (+/+ CON). From the 4th week after birth, the rats in the RMA treatment groups were treated by gavage with the designated doses of drug for 12 weeks; the rats in the control group received by gavage the same volume of 0.5% sodium CMC/ saline solution. Rats were given unrestricted access to food and water. Blood and urine samples were collected from the RMA- and vehicle-treated rats after 4, 8 and 12 weeks of treatment. Kidneys were harvested (half of each kidney was snapfrozen and the other half was fixed in 4% paraformaldehyde for subsequent morphological analysis) after 12 weeks of treatment. All animal experiments and the collection of human samples were approved by the Ethics Committee of the Second Military Medical University, Shanghai, China. Detection of biochemical indices and renal function The Synchron LX, UniCel DxC 600/800 and Synchron AQUA CAL 1 and 2 Systems were used for quantitative determination of creatinine, blood urea nitrogen (BUN), alanine aminotransferase, aspartate aminotransferase and lipid concentrations in plasma and urine of rats and mice.
Complement pathway and ADPKD
Urinary alternative pathway haemolytic complement activity (AH50) was measured using the rat AH50 enzyme-linked immunosorbent assay kit (Groundwork Biotechnology Diagnostics, Ltd, San Diego, CA, USA) according to the manufacturer’s instructions. Immunohistochemical analysis Complement Paraffin sections (3 lm thick) were cut from renal tissue blocks and incubated with a primary antibody against CFB (1 : 300, R&D systems), C9 (1 : 100, Abcam) or C5b-9 (1 : 200, Sigma-Aldrich) at 4 °C overnight and stained with chromogen 3,3diaminobenzidine (DAB) using the ABC kit (Thermo Scientific, San Jose, CA, USA) according to the manufacturer’s instructions. Ki67 Immunohistochemical staining for Ki67 was performed on 3-lm-thick tissue sections. In brief, the tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed in an autoclave oven. A mouse anti-Ki67 antibody (1 : 600, Abcam) was used as the primary antibody. After applying the primary antibody for 1 h, the sections were washed and then incubated with biotinylated secondary antibody (Vector) for 30 min. This was followed by application of the ABC reagent (Vector). Diaminobenzidine with metal enhancement was used as the detection reagent. For each section, we randomly chose 10 cysts, counted the number of Ki67-positive nuclei and then averaged the number of Ki67-positive nuclei per cyst. Quantification of kidney morphology Six representative images (1009 magnification) stained with haematoxylin and eosin were captured from renal sections of every animal from each group. The images were resized to 800 9 598 pixels, a 5 9 5 grid was placed over each image, and each square counts as one field. The infiltration of inflammatory cells was counted manually and expressed as mean number of cells per 25 fields for each sample. Periodic Acid Schiff (PAS)stained sections were subjected to cyst index analysis, using HistoQuest image analysis software (TissueGnostics, Vienna, Austria), to count the entire cortical region [total area (TA)] and the cyst area (CA) in the renal cortex. The cyst index was calculated as CA/TA 9 100. The interstitial fibrosis index was also measured in the cortical region ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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of PAS-stained sections. The fibrosis index was calculated as the positive pink–purple area/ TA 9 100. All analyses were performed by a na€ıve observer using the HistoQuest software. Statistical analysis Data are presented as means SD. Statistical differences between treatment groups were determined by one-way ANOVA followed by Bonferroni’s test. P < 0.05 was considered to be statistically significant. Results Analysis of urinary glycoproteome in normal control subjects and ADPKD patients: complement components and related proteins We initially hypothesized that complement components in the urinary glycoproteome might serve as important biomarkers for ADPKD. Patients were divided into three groups based on CKD stage: ADPKD stage 1 (GFR ≥ 90 mL min 1 per 1.73 m2), ADPKD stage 2–3 (GFR 30–89 mL min 1 per 1.73 m2) and ADPKD stage 4-5 (GFR ≤ 29 mL min 1 per 1.73 m2); a fourth group of healthy individuals served as the control (Table 1). Ten urine samples from each group were pooled and analysed. We identified a total of 113 different glycoproteins in the urine of ADPKD patients. Details regarding these glycoproteins are provided in the online Supplement (Tables S1–S3 and Figures S1–S3). Of the 113 glycoproteins, there were six complement and complement-related proteins: CFB, SERPING1, C9, C1RL, CD55 and CD59 (Table S4). We then analysed the relative differences in these complement proteins in the urine between controls and ADPKD patients at different CKD stages. For each group, a minimum increase or decrease of 50% (i.e. ≥1.5 or ≤0.67, respectively) compared with controls or between ADPKD stages was considered significant [11]. The levels of CFB, SERPING1 and C9 were increased, whereas the levels of C1RL, CD55 and CD59 were decreased with ADPKD progression (Table 2). Independent validation of changes in urinary complement proteins in ADPKD patients The identification of the aforementioned six complement-related proteins by mass spectroscopy was validation again, because these six proteins were identified as multiple peptides ranging from 12 to 26 peptides for each complement component (Table S4). Nonetheless, it is well known that 474
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changes in glycoprotein levels do not necessarily correlate with the intact proteins that are measured by immunological tests. Thus, we also quantified these six complement-related proteins in the urine using Western blot analysis (Fig. 1) and found that the levels of CFB, SERPING1 and C9 were increased, whereas C1RL, CD55 and CD59 levels were decreased with advancing ADPKD stage. All candidate bands detected by western blotting were then digested and subjected to MS analysis, which confirmed that the bands represented the proteins of interest (Table S5, Figures S2 and S3). Collectively, our data indicate that changes in these six complement proteins are reliably associated with progression of ADPKD. Serum levels of C3 and C4 are not altered in ADPKD patients To determine whether the changes in urinary complement proteins were specifically correlated with the activation of the complement pathway in the kidney or whether they derived from filtered complement components from the blood, we measured the serum levels of C3 and C4, two complement components that are routinely used to monitor systemic complement activity. We found that serum C3 and C4 levels in ADPKD patients were not significantly different from those in normal control subjects (Table 1). These results argue against the possibility of systemic complement activation and suggest that the observed changes in urinary complement components are due to activation of the complement pathway within the kidney. Expression of CFB and C9 in cyst-lining epithelial cells from ADPKD patients and in renal tubular cells from patients with other types of CKD To test the hypothesis that the alternative complement pathway is over-activated in the kidneys of ADPKD patients, we analysed the expression of CFB and C9 in human kidney tissues using immunohistochemistry. Robust CFB signals were detected in the renal tubular and cyst-lining epithelial cells from ADPKD patients, and a few positive signals were observed in renal tubular cells from patients with diabetic nephropathy (DN) and IgAN, whereas no positive signal was detected in the renal tubular of patients with minimal change disease (MCD) or LN, or in normal control subjects. C9 signals were significantly increased in the cyst-lining epithelial cells from ADPKD patients, with minor increases in tubular cells from IgAN and DN patients, but no expression in patients with MCD or LN or in control subjects (Fig. 2a).
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Table 2 Complement-related protein expression in urine samples from ADPKD patients and control subjects as analysed by liquid chromatography–mass spectrometry ADPKD
ADPKD
ADPKD
Accession
stage 1 vs.
stages 2–3
stages 4–5
Names
number
control
vs. control
vs. control
CFB Complement factor B
IPI00639937.1
1.1
1.56
4.71
5.17
CFB Isoform 1 of Complement
IPI00019591.1
factor B precursor Fragment C9 Complement component C9 precursor
IPI00022395.1
1.17
2.77
SERPING1 Complement component 1 inhibitor precursor
IPI00879931.1
1.23
1.49
1.54
C1RL 48 kDa protein
IPI00872573.1
1.4
0.94
0.63
0.65
0.70
0.57
1.06
0.98
0.61
C1RL cDNA FLJ14022 fis, clone HEMBA1003538
IPI00795055.1
weakly similar to COMPLEMENT C1R COMPONENT C1RL Complement C1r subcomponent-like protein precursor
IPI00009793.4
CD55 Decay-accelerating factor splicing variant 5
IPI00382926.2
CD55 Decay-accelerating factor splicing variant 1
IPI00784169.1
CD59 glycoprotein precursor
IPI00011302.1
Data are presented as ratios, which are the stages of autosomal dominant polycystic kidney disease (ADPKD) group divided by the control group respectively. Ratios of ≥1.5 and ≤0.67 denote significant differences from control. IPI, international protein index.
Western blot analysis results further confirmed the upregulation of CFB and C9 in the kidney tissues from ADPKD patients (Fig. 2b). These results demonstrate that the alternative pathway is clearly overactivated in renal cyst tissues of ADPKD patients. Elevated levels of C9 in cyst-lining epithelial cells from conditional Pkd1 / knockout mice To probe whether the complement system is actively involved in the initiation of ADPKD or contributes to its progression, an animal model is required. As an initial step, we measured C9 expression in an Mx1Cre+Pkd1null/flox mouse model [12]. After administration of the interferon inducer polyinosinicpolycytidylic acid (62.5 lg) for five consecutive days at P7 by intraperitoneal injection, kidneys were harvested at 11 weeks of age. No significant difference in C9 expression between normal and precystic kidney tissues was found with immunofluorescence staining, whereas C9 immunoreactivity was highly positive in the cyst-lining epithelial cells of cystic kidney tissues (Fig. 2c). Pharmacological inhibition of the complement pathway attenuates disease progression in conditional Pkd1 / knockout mice We then examined the effect of the complement system inhibitor RMA on disease progression in
conditional Pkd1 / mice. After 4 weeks (P7 to P35) of RMA (300 mg kg 1 day 1) treatment (Fig. 3a), BUN was significantly decreased in RMA-treated Pkd1 / mice (9.6 1.3 mmol L 1) compared with vehicle-treated Pkd1 / animals (43.2 7.8 mmol L 1); the same effect was observed for plasma creatinine levels (17.4 1.1 lmol L 1 vs. 35.8 9.1 lmol L 1; Fig. 3b). In RMA-treated Pkd1 / mice, the cyst index was reduced by 59.8% and the two kidneys/body weight ratio (2K/ BW%) was reduced by 60.3%, compared with vehicle-treated Pkd1 / mice, without any body weight loss (Fig. 3b and c). Complement factor B and C5b-9 expressions in renal tissues from Pkd1 / mice were upregulated compared with Pkd1+/+ mice. RMA treatment reduced CFB and C5b-9 expression in Pkd1 / mice compared with vehicle-treated Pkd1 / animals (Fig. 3d). Pharmacological inhibition of the complement pathway attenuates disease progression in Han:SPRD rats Compared with the vehicle-treated control animals, BUN and plasma creatinine levels were significantly decreased in rats treated with different dosages of RMA (150 or 300 mg kg 1 /day 1; Fig. 4a), indicating that RMA treatment improved kidney function in Han:SPRD Cy/+ rats. The 2K/BW ratio and cystic index were significantly improved in ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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Control
(a)
ADPKD stage 2~3
ADPKD stage 1
ADPKD stage 4~5
CFB
C9
SERPING1
C1RL
CD55
CD59
CFB
(b)
C9
3500
*
3000 2500 2000
*
2500 2000 1500
1500 1000 500 0
1000 500 0
Control
ADPKD stage 1
ADPKD ADPKD stage 2–3 stage 4–5
Control
3000
*
2500 2000 1500 1000 500
Control
ADPKD stage 1
ADPKD ADPKD stage 2–3 stage 4–5
C1RL
SERPING1
0
ADPKD stage 1
ADPKD ADPKD stage 2–3 stage 4–5
1800 1600 1400 1200 1000 800 600 400 200 0
* Control
CD55
ADPKD stage 1
ADPKD ADPKD stage 2–3 stage 4–5
CD59 2500
1400 1200 1000 800 600 400
2000 1500
*
200 0
Control
ADPKD stage 1
*
ADPKD ADPKD stage 2–3 stage 4–5
*
1000 500 0
Control
ADPKD stage 1
ADPKD ADPKD stage 2–3 stage 4–5
Fig. 1 Western blotting (a) and densitometric (b) analysis of complement-related protein expression in urine samples from autosomal dominant polycystic kidney disease (ADPKD) patients and control subjects. The protein expression levels of complement factor B (CFB), C9 and SERPING1 were increased, whereas those of C1RL, CD55 and CD59 were decreased with ADPKD progression. 476
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(a)
(b)
(c)
Fig. 2 Immunohistochemical staining (a) and Western blotting and densitometric analysis (b) of complement factor B (CFB) and C9 in the kidney of autosomal dominant polycystic kidney disease (ADPKD) patients and immunofluorescence staining of C9 in normal, precystic and cystic kidney tissues from conditional Pkd1 / knockout mice (c). No positive signal for either CFB or C9 was detected in the renal cortex of normal control subjects or of MCN and LN patients, whereas slight CFB and C9 signals were detected in DN and IgAN patients, with robust signals in the renal tubular and cyst epithelial cells of ADPKD patients (a). Compared with normal control subjects, the protein expression levels of both CFB and C9 in the kidney tissues from ADPKD patients were significantly increased (*P < 0.05 vs. normal control subjects; b). There was a significant upregulation of C9 in cystic kidney compared to normal kidney; C9 was mainly localized to cyst-lining epithelial cells (c). MCD, minimal change disease, LN, lupus nephritis; DN, diabetic nephropathy; IgAN, IgA nephropathy.
RMA-treated Han:SPRD Cy/+ rats with either dose (P < 0.05) compared with the control group after 12 weeks of treatment (Fig. 4b,c). Using immunohistochemistry, we also found robust positive signals for CFB and C5b-9 in the renal tubular and cyst-lining epithelial cells in vehicle-treated Cy/+ rats, which were significantly inhibited by RMA
treatment; these complement proteins were not detectable in Han:SPRD +/+ rats (Fig. 4d). To confirm the efficacy of RMA in inhibiting complement activation, we measured the haemolytic activity (AH50) of the complement pathway in rat urine after 12 weeks of treatment. We found that ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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(a)
(b)
Fig. 3 Therapeutic effect of rosmarinic acid (RMA) in conditional Pkd1 / knockout mice. Schematic diagram of the experimental design (a); blood urea nitrogen (BUN), serum creatinine (sCr), body weight, total kidney weight, two kidneys/ body weight ratio (2K/BW%) and cyst index after 4 weeks of RMA treatment (b); Periodic Acid Schiff (PAS) staining of kidney sections (c); renal expression of complement factor B (CFB) and C5b-9 by immunohistochemical staining (d). RMA treatment significantly reduced BUN, sCr, total kidney weight, 2K/BW% and cyst index without affecting body weight. Expression of CFB and C5b-9 was inhibited after RMA treatment.
the AH50 activity in the urine was significantly lower in Han:SPRD Cy/+ rats treated with RMA than in vehicle-treated animals (Table S6). In addition, there were no significant differences in liver function or triglyceride and cholesterol levels between the control and RMA-treated groups (Table S6), indicating that RMA treatment did not have any severe side effects on either liver function or lipid metabolism. Antiproliferation, inhibition of inflammatory cell infiltration and reduced fibrosis by inhibition of the complement system in conditional Pkd1 / mice and Han:SPRD rats To explore how inhibition of the complement system may retard cystic disease progression, we determined the effect of RMA on tubular epithelial cell proliferation, tubular interstitial inflammatory cell infiltration and fibrosis. The number of Ki-67478
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positive nuclei was significantly increased in the cyst-lining epithelial cells in kidney tissues from Pkd1 / mice and Han:SPRD Cy/+ rats compared with tubular epithelial cells in kidney tissues from Pkd1+/+ mice and Han:SPRD +/+ rats. RMA treatment led to a significant reduction in the number of Ki-67-positive nuclei in cyst-lining epithelial cells in Pkd1 / mice and Han:SPRD Cy/+ rats compared with vehicle-treated animals (Fig. 5a and Table 3), indicating that inhibition of the complement pathway is linked to a decrease in the proliferation of cyst-lining epithelial cells. Inflammatory cell infiltration in the renal interstitial area was greatly increased in kidneys from Pkd1 / mice and Han:SPRD Cy/+ rats compared with those from Pkd1+/+ mice and Han:SPRD +/+ rats and was significantly decreased after RMA treatment compared with vehicle treatment in
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(c)
(d)
Fig. 3 Continued.
Pkd1 / mice and Han:SPRD Cy/+ rats (Fig. 5b and Table 3); thus, inhibition of the complement pathway with RMA ameliorated the inflammatory cell infiltration in tubular interstitium. Renal fibrosis was assessed by PAS staining. There was a much larger amount of PAS-positive staining in the interstitial area of kidney tissues from
Pkd1 / mice and Han:SPRD Cy/+ rats compared with Pkd1+/+ mice and Han:SPRD +/+ rats. Similarly, the area between cysts and tubules was much larger in Pkd1 / mice and Han:SPRD Cy/+ rats than in Pkd1+/+ mice and Han:SPRD +/+ rats. After RMA treatment, the amount of positive PAS staining was decreased and the area between cysts and tubules became smaller in Pkd1 / mice and ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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(a)
(b)
Fig. 4 Therapeutic effect of rosmarinic acid (RMA) in Han:SPRD rats. Schematic diagram of the experimental design (a); blood urea nitrogen (BUN), serum creatinine (sCr), body weight, total kidney weight, two kidneys/body weight ratio (2K/BW %) and cyst index after 12 weeks of RMA treatment (b); Masson staining of kidney sections (c); renal expression of complement factor B (CFB), C9 and C5b-9 by immunohistochemical staining (d). RMA treatment significantly reduced BUN, sCr, total kidney weight, 2K/BW% and cyst index without affecting body weight. CFB, C9 and C5b-9 expression were inhibited after RMA treatment. Magnification: 4009 in C; scale bar: 100 lm in D. CMC-Na, sodium carboxymethylcellulose; R150, 150 mg kg 1 day 1 RMA; R300, 300 mg kg 1 day 1 RMA; CON, control.
Han:SPRD Cy/+ rats, demonstrating that inhibition of the complement system with RMA was associated with a decrease in fibrosis (Fig. 5c and Table 3). Discussion The results of the present study show that excessive activation of the alternative complement pathway is associated with ADPKD progression and that treatment with the complement inhibitor RMA had a beneficial effect on disease progression in experimental models of polycystic kidney disease. Previous studies have already demonstrated that 480
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mRNA transcripts for components of the complement system are over-expressed in kidney tissues from Han:SPRD rats [5]. Furthermore, it has been shown that substantial amounts of complement proteins are present in renal cyst fluid of ADPKD patients [6]. These preliminary findings have prompted the following questions: (i) Does complement activation occur within the kidney parenchyma of ADPKD patients? (ii) Which specific pathways of the complement system are involved? (iii) Is activation of the complement system specific for ADPKD? (iv) Is the over-expression of complement components an initial event or a sustained influence on ADPKD progression? and
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(c)
(d)
Fig. 4 Continued.
(v) finally, what are the mechanisms by which over-activation of the complement system causes ADPKD progression? To exclude the possibility that the changes in urinary complement components are not the result
of systemic complement activation, we measured serum C3 and C4 levels in ADPKD patients and normal control subjects. As serum C3 and C4 levels were normal, it appears that the changes in complement components in ADPKD occur within the kidney. Furthermore, the 24-h urinary protein ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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Table 3 Effect of rosmarinic acid treatment on Ki-67 immunohistochemistry staining, inflammatory cell infiltration and fibrosis in kidney tissues from conditional Pkd1 / knockout mice and Han:SRPD rat Ki-67 positive nuclear
Interstitial inflammatory cells
Fibrosis index (%)
Conditional Pkd1 knock-out mice Pkd1
/
R300
1.1 0.1***
Pkd1
/
CON
5.9 0.3
Pkd1+/+ R300 +/+
Pkd1
CON
6.4 0.3** 12.5 1.4
3.9 1.4** 12.3 1.7
0.5 0.1***
1.1 0.2***
1.6 0.9***
0.5 0.1***
1.2 0.2***
3.8 1.0***
Han:SPRD rat Cy/+ R300
1.7 0.5^^
23.8 1.8^^
Cy/+ CON
2.6 0.7
42.2 1.6
+/+ R300
1.0 0.1
4.6 0.1
6.3 1.3^^^
+/+ CON
0.9 0.0
4.3 0.1
7.5 2.1^^^
^^^
**P < 0.01, ***P < 0.001 vs. Pkd1
^^^
/
Using quantitative urinary glycoproteomics and various confirmatory techniques, we have shown for the first time that the levels of the specific complement components CFB, SERPING1 and C9 are increased, whereas C1RL, CD55 and CD59 levels are decreased in urine samples from ADPKD patients. SERPING1 is one of the key regulators of the complement pathways, strongly inhibiting C1r and C1s in the classical pathway as well as MASP-1 and MASP-2 in the MBL pathway [13]. Thus, SERPING1 not only controls the initiation of the classical pathway but also inhibits the early activation steps of the MBL pathway. The increase in SERPING1 found in the present study suggests that these two pathways are unlikely to be activated in ADPKD patients. Complement factor B is a crucial factor for initiating and sustaining the activation of the alternative pathway [14]. CD55 is an inhibitor of C3 convertase, C9 participates in the formation of C5b-9, and CD59 inhibits the function of C9. The decreases in CD55 and CD59 are conducive to the activation of the complement pathway (Fig. 6). Therefore, the observed alterations in the levels of these six complement-related proteins in the urine of ADPKD patients are consistent with the selective activation of the alternative complement pathway. ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
^^^
17.7 5.3
CON group; ^^P < 0.01, ^^^P < 0.001 vs Han:SPRD Cy/+ CON group.
excretion was within normal limits in all groups of ADPKD patients (Table 1), suggesting again that the complement proteins present in the urine of ADPKD patients are not the result of leakage through the glomerular barrier, but rather originate from renal tubular cells and cyst-lining epithelial cells.
482
^^^
7.9 3.9^^
Complement factor B is a crucial factor for initiating and sustaining the activation of the alternative complement pathway, and C9 is important for the formation of the final membrane attacking complex C5b-9; therefore, next we determined the levels of CFB and C9 in kidney tissues of patients with ADPKD and compared them to levels in patients with other types of chronic kidney disease (MCD, DN, IgAN and LN). Immunohistochemical staining revealed that CFB and C9 were markedly overexpressed in tubular and cyst-lining epithelial cells in kidney tissues from ADPKD patients, but only slightly increased in renal tubular cells of kidney biopsy samples from DN and IgAN patients. In addition, no staining was observed in patients with MCD and LN. These findings suggest that the overexpression of CFB and C9 is specific to patients with ADPKD. We also found that the increases in urinary complement components CFB, SERPING1 and C9 and the decreases in C1RL, CD55 and CD59 were correlated with the different stages of ADPKD. C9 is not expressed in normal and precystic kidneys, but is highly expressed in the cyst-lining epithelial cells of cystic kidney tissues. We therefore investigated the effect of RMA which has been reported to inhibit complement activation predominately by reacting covalently with the activated thioester in nascent C3b, thus blocking C3b attachment to complement-activating surfaces. We found that RMA inhibited the expression of CFB and C9, reduced cyst index and 2K/BW% and improved renal function in conditional Pkd1 / knockout mice and Han:SPRD Cy/+ rats. These data strongly
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(a)
(b)
Fig. 5 Mechanism of complement-mediated progression of autosomal dominant polycystic kidney disease (ADPKD). Ki-67 immunohistochemical staining (a). The Ki-67-positive signal was black in Pkd1 mice and dark brown in Han:SRPD rats due to the different visualization reagents. Inflammatory cell infiltration determined in haematoxylin-and-eosin-stained sections (b) and fibrosis visualised by PAS staining (c). The PAS-positive signal was pink– purple. R300, 300 mg kg 1 day 1 RMA, rosmarinic acid; CON, control.
(c)
suggest that over-activation of the alternative complement pathway is important in the progression of ADPKD, rather than an as an inducer of cystogenesis. The mechanisms that lead to over-activation of the alternative complement pathway in ADPKD are complex. The number of Ki-67-positive cells and interstitial inflammatory cell infiltration and the
degree of interstitial fibrosis were all reduced by RMA treatment in kidney tissues from conditional Pkd1 / knockout mice and Han:SPRD Cy/+ rats, indicating that ADPKD progression which is mediated by the over-activation of the alternative complement pathway is due at least in part to proliferation of cyst-lining epithelial cells, interstitial inflammatory cell infiltration and interstitial fibrosis. ª 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2014, 276; 470–485
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urinary glycoproteins and achieved an enrichment of these low-abundance proteins with the use of hydrazide beads. This approach allowed enrichment of glycoproteins and reduction in the overall range of proteins. Amongst 139 identified proteins, a total of 113 were genuine glycoproteins, which reflects a specificity of 81.3%. To the best of our knowledge, our study is the first comprehensive and systematic characterization of the urinary glycoproteome (Table S1). In a few previous studies, the levels of total, membrane and phosphorylated proteins have been analysed in urine with or without fractionation [17, 18]. Such unbiased profiling tends to preferentially identify more abundant proteins. In our study, 21 of the 261 glycopeptides have not been annotated by the current database (Table S3).
Fig. 6 Schematic diagram of excessive activation of the alternative complement pathway in autosomal dominant polycystic kidney disease (ADPKD) progression. Red denotes upregulation; green denotes downregulation; ? denotes stimulation; denotes inhibition; pink rectangle denotes the effect of rosmarinic acid (RMA). CFB, complement factor B.
A limitation of this study is that RMA is not a specific complement inhibitor, as it also inhibits glutathione reductase, glucose-6-phosphate dehydrogenase and the caspase-1 pathway. A more specific inhibitor of the alternative complement pathway or the cross-breeding between CFB knockout mice and Pkd1 knockout mice would be required to confirm the unique role of the alternative complement pathway in ADPKD progression. Nevertheless, our findings provide hope that inhibitors of the same type as RMA could be useful agents to retard disease progression in patients with ADPKD at least in part by blocking the alternative complement system. It is worth mentioning a major technical obstacle to the identification of proteins by proteomic methodologies in body fluids with a vast array of proteins (including plasma and urine): mass spectrometry is typically biased towards abundant proteins [15]. Several approaches have been employed to overcome this problem [16]. In the present study, we focused on the subproteome of 484
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In conclusion, using a robust quantitative proteomics screen, we identified 113 different glycoproteins and 261 glycopeptides in the urine; moreover, we found significant expression changes in six complement components in the urine from ADPKD patients. Excessive activation of the alternative complement pathway was observed in cystic kidneys of ADPKD patients, conditional Pkd1 / mice and Han:SPRD rats and may exacerbate ADPKD progression. The complement inhibitor RMA effectively ameliorated the pathological changes and delayed the deterioration of renal function in conditional Pkd1 / mice and Han:SPRD rats, which suggests that targeting the complement system may be a novel therapeutic strategy for ADPKD. Conflict of interest statement The authors declare that they have no competing interests. Acknowledgements This work was supported by the National High Technology Research and Development Program 863 Project (2007AA02Z3Z1), National Natural Science Foundation of China General Projects (30971368, 30871179), Shanghai Key Discipline Project (B902), Major National Science and Technology Project (2009ZX09102), Shanghai Science and Technology Commission Major Research Project (11431920800), Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents, Qianjiang Talents Project of Science and Technology Department in Zhejiang Province, Shanghai International Science and Technology
Z. Su et al.
Cooperation Fund Project (0954070200) to CM; the Shanghai Science and Technology Committee General Project (09ZR1410400) to XW; National Institute of Child Health and Human Development (P30 HD02274) to RPB; and the Swiss National Science Foundation (320030-144093 to RPW and 310030132597 to ALS).
References 1 The International Polycystic Kidney Disease Consortium. Polycystic kidney disease: the complete structure of the PKD1 gene and its protein. The International Polycystic Kidney Disease Consortium. Cell 1995; 81: 289–98. 2 Mochizuki T, Wu G, Hayashi T et al. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science 1996; 272: 1339–42. 3 Patel V, Chowdhury R, Igarashi P. Advances in the pathogenesis and treatment of polycystic kidney disease. Curr Opin Nephrol Hypertens 2009; 18: 99–106. 4 Wuthrich RP, Mei C. Aquaretic treatment in polycystic kidney disease. N Engl J Med 2012; 367: 2440–2. 5 Burtey SJ, Riera M, Fontes M. Overexpression of complement-component genes in Han:SPRD rats a model of polycystic kidney disease. Kidney Int 2008; 73: 1324–5; author reply 5. 6 Lai X, Bacallao RL, Blazer-Yost BL, Hong D, Mason SB, Witzmann FA. Characterization of the renal cyst fluid proteome in autosomal dominant polycystic kidney disease (ADPKD) patients. Proteomics Clin Appl 2008; 2: 1140–52. 7 Pei Y, Obaji J, Dupuis A et al. Unified criteria for ultrasonographic diagnosis of ADPKD. J Am Soc Nephrol 2009; 20: 205–12. 8 Shi M, Bradner J, Bammler TK et al. Identification of glutathione S-transferase pi as a protein involved in Parkinson disease progression. Am J Pathol 2009; 175: 54–65. 9 Hong Z, Shi M, Chung KA et al. DJ-1 and alpha-synuclein in human cerebrospinal fluid as biomarkers of Parkinson’s disease. Brain 2010; 133: 713–26. 10 Piontek KB, Huso DL, Grinberg A et al. A functional floxed allele of Pkd1 that can be conditionally inactivated in vivo. J Am Soc Nephrol 2004; 15: 3035–43. 11 Zhang J, Goodlett DR, Peskind ER et al. Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid. Neurobiol Aging 2005; 26: 207–27. 12 Takakura A, Contrino L, Beck AW, Zhou J. Pkd1 inactivation induced in adulthood produces focal cystic disease. J Am Soc Nephrol 2008; 19: 2351–63. 13 Cugno M, Zanichelli A, Foieni F, Caccia S, Cicardi M. C1-inhibitor deficiency and angioedema: molecular mechanisms and clinical progress. Trends Mol Med 2009; 15: 69–78. 14 Le Quintrec M, Lionet A, Kamar N et al. Complement mutation-associated de novo thrombotic microangiopathy following kidney transplantation. Am J Transplant 2008; 8: 1694–701.
Complement pathway and ADPKD
15 Wang L, Li F, Sun W et al. Concanavalin A-captured glycoproteins in healthy human urine. Mol Cell Proteomics 2006; 5: 560–2. 16 Ghosh D, Krokhin O, Antonovici M et al. Lectin affinity as an approach to the proteomic analysis of membrane glycoproteins. J Proteome Res 2004; 3: 841–50. 17 Li QR, Fan KX, Li RX et al. A comprehensive and non-prefractionation on the protein level approach for the human urinary proteome: touching phosphorylation in urine. Rapid Commun Mass Spectrom 2010; 24: 823–32. 18 Lai ZW, Steer DL, Smith AI. Membrane proteomics: the development of diagnostics based on protein shedding. Curr Opin Mol Ther 2009; 11: 623–31. Correspondence: Prof. Changlin Mei, Kidney Institute, Department of Nephrology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, China. (Fax: (0086)-21-63521416; e-mail: [email protected]). Prof. Rudolf P. W€ uthrich, Division of Nephrology, University Hospital Zurich, University of Zurich, 8091 Zurich, Switzerland. (Fax: (0041)-44-2554593; e-mail: [email protected])
Supporting Information Additional Supporting Information may be found in the online version of this article: FigureS1. Gene ontology analysis of glycoprotein identified in human ADPKD urine. FigureS2. Characterization of CFBin human urine. Figure S3. Characterization of SERPING1, C9, C1RL, CD55 and CD59in human urine. Table S1. Glycoproteins and Glycopeptides Identified in Human Urine. Table S2. Glucoproteins identified in human urine of previous investigations. Table S3. Glycopeptides unannotated by current database. Table S4. The 6 complement-related proteins identified by multiple peptides. Table S5. Human CFB,SERPING1,C9,C1RL,CD55 and CD59peptides identified by mass spectrometry in urine.
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