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【摘要】 Transforming growth factor- 1 (TGF- 1 ) is not only an important fibrogenic but also immunomodulatory cytokine in the human kidney. We have recently demonstrated that TGF- 1 induces interleukin-8 (IL-8), macrophage chemoattractant protein-1 (MCP-1), and fibronectin production in renal proximal tubular (HK-2) cells. However, the unique dependence of IL-8, MCP-1, and fibronectin on TGF- 1 expression is unknown. The TGF- 1 gene was effectively silenced in HK-2 cells using small-interference (si) RNA. Basal secretion of IL-8 and MCP-1 decreased (both P < 0.05) but, paradoxically, fibronectin increased ( P < 0.05) in TGF- 1 -silenced cells compared with cells transfected with nonspecific siRNA. Significant increases were observed in mRNA for the TGF- 2 ( P < 0.05), TGF- 3 ( P < 0.05) isoforms and pSmad2 ( P < 0.05), which were reflected in protein expression. Concurrent exposure to pan-specific TGF- antibody reversed the observed increase in fibronectin expression, suggesting that TGF- 2 and TGF- 3 isoforms mediate the increased fibronectin expression in TGF- 1 -silenced cells. An increase in the DNA binding activity of activator protein-1 (AP-1; P < 0.05) was also observed in TGF- 1 -silenced cells. In contrast, nuclear factor- B (NF- B) DNA binding activity was significantly decreased ( P < 0.0005). These studies demonstrate that TGF- 1 is a key regulator of IL-8 and MCP-1, whereas fibronectin expression is regulated by a complex interaction between the TGF- isoforms in the HK-2 proximal tubular cell line. Decreased expression of TGF- 1 reduces chemokine production in association with reduced NF- B DNA binding activity, suggesting that immunomodulatory pathways in the kidney are specifically dependent on TGF- 1. Conversely, decreased expression of TGF- 1 results in increased TGF- 2, TGF- 3, AP-1, and pSmad2 that potentially mediates the observed increase in fibronectin.
【关键词】 small interference RNA chemokine pSmad activator protein nuclear factor B
TRANSFORMING GROWTH FACTOR (TGF)- is known to be a multifunctional peptide. The functions of TGF- can be grouped into three broad areas: modulation of inflammatory cell function, growth inhibition and differentiation, and control of extracellular matrix production. Within the kidney, TGF- 1 is recognized among the TGF- isoforms to play the most important role in renal fibrogenesis and progressive kidney disease. However, the protean and important biological functions of TGF- 1 are appreciated in TGF- 1 knockout mice, which develop diffuse mononuclear cell infiltrates that prove lethal within a few weeks of birth ( 19, 32 ). The other TGF- isoforms present in the kidney, TGF- 2 and TGF- 3, have also been shown to be fibrogenic in in vitro and in vivo models of fibrotic disease ( 10, 12, 13, 28, 39 ). In contrast, TGF- 2 and TGF- 3 knockout mice display nonlethal developmental defects ( 24, 30 ). Plasma levels of TGF- 2 and TGF- 3 are undetectable, suggesting that they are restricted to more localized autocrine and paracrine modes of action ( 2 ). We have recently demonstrated that TGF- 1 is not only an important fibrogenic cytokine but also an important regulator of chemokine production in renal tubular cells, with differential dependence on downstream production of connective tissue growth factor ( 27 ).
It is well known that TGF- signals through its receptors, TGF- type I and type II receptors, with downstream activation of the Smad signaling pathway. TGF- is known to mediate its fibrotic effects by activating the receptor-associated Smads (Smad-2 and -3). The pSmad-2 and pSmad-3 associate to form a heteromultimer with Smad-4 (Co-Smad). This complex is then translocated to the nucleus, where it can regulate target gene expression ( 31 ).
Many lines of evidence suggest nuclear transcription factors AP-1 and NF- B play regulatory roles in TGF- 1 -dependent extracellular matrix and chemokine production ( 1, 3, 7, 20, 34, 37 ). IL-8, MCP-1, and fibronectin promoters contain both AP-1 and NF- B binding sites and, in turn, TGF- 1 regulates the activation of AP-1 and NF- B ( 21, 33, 35, 40, 41 ).
The present study aimed to determine whether the secretion of chemokines (IL-8, MCP-1) and the extracellular matrix protein fibronectin is uniquely dependent on TGF- 1 by using a renal proximal tubular cell model in which TGF- 1 is selectively silenced.
MATERIALS AND METHODS
Cell culture. The immortalized human kidney proximal tubular cell line (HK-2, ATCC) was used as the proximal tubular cell model. Cells were grown to subconfluence in keratinocyte serum-free media (Invitrogen) under conditions previously described ( 27 ).
Materials. TGF- 1 was selectively silenced in the HK-2 cells using small interference (si) RNA methodology. TGF- 1 and nonspecific double-stranded siRNA (21-mer RNA molecules) were chemically synthesized (Ambion). Human IL-8, TGF- 2 ELISA kits, pan-specific TGF- antibody, and rabbit IgG were all purchased from R&D Systems (Minneapolis, MN), a human MCP-1 immunoassay kit was from BioSource International, and a TGF- 1 ELISA kit was from Promega (Madison, WI). Antibodies to fibronectin, TGF- 3, and pSmad2 were purchased from Neomarkers (Santa Cruz, CA) and Cell Signaling (Danvers, NY), respectively.
Experimental protocol. TGF- 1 siRNA (30 nM/l) was introduced into HK-2 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer?s instructions. In parallel, cells were transfected with 30 nM/l nonspecific siRNA, which served as the control data set. RNA was extracted for verification of TGF- 1 gene silencing in the cells.
The basal levels of chemokine (IL-8 and MCP-1) and fibronectin protein in the TGF- 1 gene-silenced and control cells were determined. Media was replaced 24 h after transfection with either TGF- 1 -specific or nonspecific siRNA, and conditioned media was collected at 48 h for measurement of IL-8, MCP-1, and fibronectin. The downstream Smad-2 signaling pathway, TGF- 2 and TGF- 3 isoforms, and the TGF- -responsive transcription factors AP-1 and NF- B were assayed.
To determine whether TGF- 2 and TGF- 3 account for the increased fibronectin in TGF- 1 - silenced cells, HK-2 cells were transfected with TGF- 1 siRNA, and 5 µg/ml pan-specific TGF- antibody was added 6 h after transfection. Treatment media was replaced 24 h after transfection, and the supernatant was collected at 48 h for measurement of fibronectin protein.
siRNA. RNA molecules (21 mer) were chemically synthesized (Ambion). The complementary oligonucleotides were 2'-deprotected, annealed, and purified by the manufacturer. The sequence targeting TGF- 1 (accession no. NM_000660 ) was 5'-AAGGGCTACCATGCCAACTTC-3'.
RT-PCR. RNA was extracted using a RNeasy Mini kit (Qiagen) according to the manufacturer?s instructions. RNA was treated with DNase I (Invitrogen) and then reverse transcribed using Superscript II RT (Invitrogen). Sequence-specific primers for human TGF- 1, TGF- 2, TGF- 3, Smad2, -actin, and fibronectin are shown in Table 1. Amplified products were electrophoresed through 1.5% (wt/vol) agarose gels and visualized by ethidium bromide staining. Bands were scanned using Gel Documentation (Bio-Rad) and quantitated by densitometry using Quantity One software (Bio-Rad). -Actin was used as an internal control for sample normalization. To confirm that DNase I-treated RNA samples have no genomic DNA contamination, one negative control was always included without any Superscript II RT in reverse transcription, and subsequently this sample was used for PCR. There was no band observed on agarose gels in the negative control samples.
Table 1. Sequence-specific primers for human TGF- 1, - 2, - 3, Smad 2, -actin, and fibronectin
IL-8, MCP-1, TGF- 1, and TGF- 2 immunoassays. Conditioned media was collected and centrifuged at 3,000 rpm and 4°C for 10 min to remove cell debris and then stored at -80°C. IL-8, MCP-1, TGF- 1, and TGF- 2 were quantified using an ELISA according to the manufacturer?s instructions.
Western blotting. Conditioned media samples were subjected to SDS-PAGE under reducing conditions. Proteins were then transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech). Nonspecific binding sites were blocked overnight (5% nonfat milk and 0.1% Tween 20 in PBS) after which the membranes were incubated with primary fibronectin antibody for 2 h at room temperature, followed by washing four times, after which they were incubated with peroxidase-labeled secondary antibodies (Amersham Pharmacia Biotech) for 1 h and again washed four times. The blots were then detected using ECL (Amersham Pharmacia Biotech). The bands corresponding to fibronectin (220 kDa) were quantitated using Quantity One software (Bio-Rad). Coomassie brilliant blue staining was used to confirm that an equal amount of protein was loaded in each lane.
Immunocytochemical staining. Cells were fixed on coverslips using ice-cold 4% formalin for 30 min followed by washing three times in PBS. Cells were then permeabilized with 0.2% Triton X-100 for 20 min followed by three washings in PBS. Cells were treated with 4% H 2 O 2 in PBS to quench the endogenous peroxidase activity, and then the nonspecific background was blocked using serum-free protein block (Dako). Cells were incubated with TGF- 3 and pSmad2 primary antibodies diluted in 1:200 at room temperature for 1 h followed by three washings in PBS. LSAB2 system-horseradish peroxidase was used for detection of TGF- 3 and pSmad2 according to the manufacturer?s instructions (Dako). Mayer?s hematoxylin was used to counterstain the cells. A negative control (no primary antibody) was performed. Coverslips were mounted on glass slides using Dako glycerol mounting media. Slides were blinded, and three random fields were digitized using a Nikon microscope attached to a digital camera.
EMSA. Nuclear extract was prepared using NucBuster Protein Extraction Kit (Novagen) according to the manufacturer?s instructions. AP-1 wild-type oligonucleotide sequences were 5'-CGCTTGATGAGTCAGCCGGAA-3' and 5'-TTCCGGCTGACTCATCAAGCG-3'; and NF- B wild-type oligonucleotide sequences were 5'-CGCTTGATGACTCAGCCGGAA-3' and 5'-GCGAACTACTGAGTCGGCCTT-3'. A DIG Gel Shift Kit (Roche) was used to label AP-1 and NF- B oligonucleotide 3'-ends with DIG-11-dUTP. In brief, 40 µg of nuclear extract were used in the binding reaction and incubated at room temperature for 1 h. The binding reactions were then electrophoresed through 6% nondenaturing polyacrylamide gels and transferred to a positively charged nylon membrane (Roche). The membrane was then cross linked using a UV transilluminator for 3 min. The membrane was subjected to immunological detection using DIG-AP conjugate provided in the DIG Gel Shift Kit. Chemiluminescence was detected using ECL hyperfilm (Amersham). Both shift bands and free probes were scanned and quantitated using Gel Documentation and Quantity One (Bio-Rad). Signal percentage [shift band vs. (shift band + free probe)·100%] was calculated, and results were expressed as a percentage of control values.
Statistical analysis. All results are expressed as a percentage of the control values (100%). Each experiment was performed independently a minimum of three times. Results are expressed as means ± SE. Statistical comparisons between groups were made by ANOVA, with pairwise multiple comparisons made by Fisher?s protected least-significant difference test. Analyses were performed using the software package Statview version 4.5 (Abacus Concepts, Berkeley, CA). P values <0.05 were considered significant.
RESULTS
TGF- 1 siRNA-mediated gene silencing. TGF- 1 siRNA (30 nM/l) decreased TGF- 1 mRNA expression by 89 ± 2.8%. A RT-PCR representative gel is shown in Fig. 1 A. Total and active TGF- 1 secretion was also decreased by 66 ± 7.5 and 60 ± 0.5%, respectively ( Fig. 1, B and C ). Nonspecific siRNA is used as a control to exclude any effect caused by TGF- 1 siRNA not specifically due to silencing of TGF- 1. Transfection efficiency, estimated by transfecting HK-2 cells with green fluorescent protein plasmids (data not shown), was 85%, hence paralleled the reduction in expression of TGF- 1.
Fig. 1. Effectiveness of transforming growth factor (TGF)- 1 small-interference (si) RNA-mediated gene silencing. Nonspecific or TGF- 1 siRNAs (30 nM/l) were introduced into HK-2 cells, respectively, at subconfluence using Lipofectamine 2000. In parallel wells, to act as negative controls, cells were transfected with transfection reagent alone (Mock). Forty-eight hours after transfection, RNA was extracted. TGF- 1 mRNA levels were determined by RT-PCR. A representative RT-PCR gel is shown ( A ). Media was replaced at 24 h after transfection, and conditioned media was collected at 48 h for measurement of total ( B ) and active TGF- 1 ( C ) using ELISA. Results are means ± SE and are standardized as a percentage of control. *** P < 0.0005 vs. control. ** P < 0.005 vs. control; n = 3.
Basal secretion of IL-8 and MCP-1 decreased in TGF- 1 -silenced cells. The basal levels of IL-8 and MCP-1 in TGF- 1 -silenced cells decreased to 62.7 ± 5.4 and 74.3 ± 6.2% (both P < 0.05), respectively, compared with nonspecific control ( Fig. 2, A and B ). We have recently demonstrated that TGF- 1 upregulates IL-8 and MCP-1 through a connective tissue growth factor-independent pathway ( 27 ). These data further confirm that TGF- 1 plays a positive role in the regulation of IL-8 and MCP-1 in HK-2 cells.
Fig. 2. Basal secretion of IL-8 and MCP-1 decreased in TGF- 1 -silenced cells. Nonspecific or TGF- 1 siRNAs (30 nM/l) were introduced into HK-2 cells, respectively, at subconfluence using Lipofectamine 2000. In parallel wells, to act as negative controls, cells were transfected with transfection reagent alone (Mock). Media was replaced at 24 h after transfection, and conditioned media was collected at 48 h for measurement of IL-8 and MCP-1 using ELISA. Results are means ± SE and are standardized as a percentage of control. * P < 0.05 vs. control; n = 6.
Enhanced basal secretion of fibronectin in TGF- 1 -silenced cells. Fibronectin mRNA expression paradoxically increased (299 ± 45% vs. control; P < 0.05; Fig. 3 A ), paralleled by an increase in fibronectin protein expression (161 ± 9.6% vs. control; P < 0.05; Fig. 3 B ) in TGF- 1 -silenced cells compared with cells transfected with nonspecific siRNA.
Fig. 3. Enhanced basal secretion of fibronectin in the TGF- 1 -silenced cells. Nonspecific or TGF- 1 siRNAs (30 nM/l) were introduced into HK-2 cells, respectively, at subconfluence using Lipofectamine 2000. In parallel wells, to act as negative controls, cells were transfected with transfection reagent alone (Mock). Twenty-four hours after transfection, cells were replaced with HK-2 growth media for another 24 h, and then RNA was extracted to verify that the TGF- 1 gene was effectively silenced. A : RT-PCR for fibronectin was performed. B : conditioned media samples were used to measure fibronectin production. Results are means ± SE and are standardized as a percentage of control. * P < 0.05 vs. control; n = 6.
TGF- 1 -induced fibronectin secretion in HK-2 cells has been previously demonstrated in our laboratory ( 27 ). The enhanced mRNA and protein levels of fibronectin in TGF- 1 -silenced cells was unexpected, and the mechanisms underlying the increased matrix production were further explored.
Increased TGF- 2, TGF- 3, and pSmad2 occurs in TGF- 1 -silenced cells. mRNA levels of TGF- 2, TGF- 3, and Smad2 significantly increased to 289 ± 76, 164 ± 31, and 195 ± 32% respectively, in TGF- 1 -silenced cells compared with cells transfected with nonspecific siRNA (all P < 0.05; Fig. 4, A, C, and E ). TGF- 2 protein was significantly increased in TGF- 1 -silenced cells to 149 ± 5.7% of that observed in cells transfected with nonspecific siRNA ( P < 0.05; Fig. 4 B ). TGF- 3 and pSmad2 expression was shown to be significantly increased when assessed immunocytochemically ( Fig. 4, D and F, respectively). This supports the view that TGF- 1 plays a critical role in regulating the other TGF- isoforms in the human kidney and suggests that TGF- 1 endogenously functions to limit TGF- 2 and TGF- 3, which can also induce fibronectin production through activation of Smad2.
Fig. 4. mRNA and protein levels of TGF- 2, TGF- 3, and Smad2 in the TGF- 1 -silenced cells. Nonspecific or TGF- 1 siRNAs (30 nM/l) were introduced into HK-2 cells, respectively, at subconfluence using Lipofectamine 2000. In parallel wells, to act as negative controls, cells were transfected with transfection reagent alone (Mock). Twenty-four hours after transfection, cells were replaced with HK-2 growth media for another 24 h, and then RNA was extracted to verify that the TGF- 1 gene was effectively silenced. RT-PCR was used to measure mRNA levels of TGF- 2 ( A ), TGF- 3 ( C ), and Smad2 ( E ); ELISA was used to measure protein level of TGF- 2 ( B ). Immunocytochemistry staining was used to measure protein levels of TGF- 3 ( D ) and pSmad2 ( F ). TGF- 1 -silenced cells were either exposed to 5 µg/ml pan-specific TGF- antibody or 5 µg/ml rabbit IgG (negative control) 6 h after transfection. Treatment media was replaced at 24 h after transfection. A supernatant was collected at 48 h after transfection for measurement of fibronectin protein ( G ). *** P < 0.0005 vs. nonspecific siRNA. * P < 0.05 vs. nonspecific siRNA. ### P < 0.0005 vs. TGF- 1 siRNA plus rabbit IgG; n = 3.
Increased fibronectin returned to the control level (cells transfected with nonspecific siRNA) when TGF- 1 -silenced cells were exposed to 5 µg/ml pan-specific TGF- antibody ( Fig. 4 G ). These data further suggest that elevated levels of TGF- 2 and TGF- 3 account for the increased fibronectin in TGF- 1 -silenced cells,
DNA binding activity of AP-1 and NF- B in TGF- 1 -silenced cells. AP-1 DNA binding activity increased to 162 ± 14% ( P < 0.05) compared with cells transfected with nonspecific siRNA ( Fig. 5 A ). In contrast, NF- B DNA binding activity decreased to 18 ± 2% ( P < 0.0005) compared with cells transfected with nonspecific siRNA ( Fig. 5 B ). This suggests differential roles of these transcription factors in mediating fibrogenic and immunomodulatory responses.
Fig. 5. Differential DNA binding activity of AP-1 and NF- B in the TGF- 1 -silenced cells. Nonspecific or TGF- 1 siRNAs (30 nM/l) were introduced into HK-2 cells, respectively, at subconfluence using Lipofectamine 2000. In parallel wells, to act as negative controls, cells were transfected with transfection reagent alone (Mock). Twenty-four hours after transfection, a nuclear extract was collected for measurement of AP-1 ( A ) and NF- B ( B ) DNA binding activity using EMSA. Signal quantification of shift bands and free probes was performed and the ratio [shift band vs. (shift band + free probe)] was calculated. Results are means ± SE and are standardized as a percentage of control (nonspecific siRNA). *** P < 0.0005 vs. control. * P < 0.05 vs. control; n = 3.
DISCUSSION
The findings of the present studies reinforce the key role that TGF- plays in both profibrotic and inflammatory renal disease. However, our findings have clearly demonstrated that the inflammatory response in the kidney, leading to an upregulation of IL-8 and MCP-1, is specifically dependent on the TGF- 1 isoform and its target transcription factor NF- B. Unexpectedly, effective silencing of the TGF- 1 isoform enhanced fibronectin expression in the proximal tubule, with further investigation suggesting that this was due to an associated upregulation in TGF- 2 and - 3 isoforms with targeting of the AP-1 transcription factor and downstream activation of the Smad2 signaling pathway. To our knowledge, this is the first report to demonstrate that upregulation of IL-8 and MCP-1 is uniquely dependent on TGF- 1 and that silencing of the TGF- 1 gene results in the increased expression of TGF- 2, TGF- 3, and AP-1 DNA binding activity.
All three TGF- isoforms have been previously reported to have fibrogenic effects on renal cells. Although TGF- 1 is generally considered to be the major or predominant isoform involved in renal fibrosis, Wilson and colleagues ( 39 ) have demonstrated that all isoforms of TGF- induce fibronectin expression in rat mesangial cells and can contribute to pathological matrix accumulation in renal fibrosis repair. TGF- 2 has been reported to be dominant in the acute phase of nephropathy in streptozotocin-induced type I diabetes in the rat ( 12 ), and treatment with a specific neutralizing antibody significantly reduced renal fibrosis in this model ( 13 ). In vitro, TGF- 2 has been reported to induce extracellular matrix (fibronectin, collagen type I, collagen type IV) and play a fibrogenic role in both a human retinal pigment epithelial cell line and cultured human optic nerve head astrocytes ( 10, 28 ). TGF- 3 has also been reported to decrease matrix metalloproteinase-2 and increase tissue inhibitor of metalloproteinase-1 and collagen type I in primary lung fibroblasts ( 26 ).
Prior studies have demonstrated the interdependence of the TGF- isoforms in different models of fibrogenesis. TGF- 2 and TGF- 3 have been shown to stimulate TGF- 1 production in human fetal lung fibroblasts, rat mesangial cells, and untransformed lung and intestinal epithelial cells ( 38, 42, 43 ). This would be consistent with the present studies, as it is likely that the absence of TGF- 1 production could lead to an unregulated upregulation of TGF- 2 and - 3 driving the increase in fibronectin secretion. TGF- 1 and TGF- 2 are equally potent in the activation of Smad2 ( 11, 23 ), whereas TGF- 3 is also reported to increase the activation of Smad2 in folliculostellate cells and murine medial edge epithelium cells ( 5, 8 ). As Smad2 is known to mediate the downstream fibrogenic response of TGF-, an upregulation in the setting of enhanced fibronectin expression was not unexpected.
Our findings are consistent with the phenotypic observations in the rare clinical disorder Camurati-Engelmann disease, characterized by unregulated excessive fibrosis of skin, subcutaneous tissue, and organs and progressive diaphyseal dysplasia characterized by hyperosteosis and sclerosis of the diaphyses of long bones ( 16 ). Both activating and inactivating mutations of several components of the TGF- signaling pathway have been described, resulting in the same clinical phenotype ( 4, 14, 29 ). For example, Kinoshita et al. ( 16 ) hypothesize that domain-specific heterozygous missense mutations in TGF- 1, with resultant inactive protein, is a likely cause of Camurati-Engelmann disease.
Conversely, in experimental models, Chen et al. ( 6 ) reported that fibronectin expression decreased in cultured tubular cells derived from TGF- 1 knockout mice. Interestingly, in these studies a significant upregulation of TGF- 3 expression was also observed, as was the expression of the type II receptor. However, constitutive expression of fibronectin was reduced. The explanation for these somewhat divergent results remains unclear.
In contrast to the paradoxical increase in fibronectin expression in the TGF- 1 -silenced cells, the production of the inflammatory chemokines IL-8 and MCP-1 was reduced. We have recently demonstrated that TGF- 1 upregulates IL-8 and MCP-1 expression in proximal tubular epithelial cells ( 27 ). Hence, these results are consistent with the recognition that TGF- 1 plays a central role in the modulation of inflammation in the tubular epithelium.
The differential effects of the effective silencing of TGF- 1 on cytokines involved in the inflammatory pathways and proteins contributing to extracellular matrix production may be explained by the differential effects on the transcription factors AP-1 and NF- B. Although both AP-1 and NF- B have been reported to be involved in the regulation of renal inflammatory responses ( 25, 36 ), AP-1 activation has been more closely linked with renal fibrosis and NF- B with inflammatory responses. TGF- 1 enhances both AP-1 and NF- B DNA binding activity ( 9, 17, 18 ). Conversely, TGF- 2 has been shown to repress NF- B activity in granule neurons ( 15 ) and TGF- 3 has been shown to induce AP-1 in untransformed lung and intestinal epithelial cells ( 43 ). Although these findings are not uniformly observed ( 9, 22 ), our findings are consistent with these observations that AP-1 and NF- B DNA binding activity and activation are differentially regulated by different isoforms of TGF-, resulting in divergent effects on the expression of potential participants in fibrotic and inflammatory pathways.
In summary, our studies in this in vitro model of tubular cells suggest that TGF- 1 is specifically involved in the modification of cytokines potentially involved in inflammatory responses, but fibronectin expression is regulated by a complex interaction between the TGF- isoforms and their interacting transcription factors and downstream signaling pathways. A key role of TGF- 1 in the proximal tubular cells of human kidney, identified in the present study, is to limit the production of the extracellular matrix protein fibronectin, induced by TGF- 2 and - 3.
GRANTS
These studies were supported by the National Health and Medical Research Council of Australia and the Juvenile Diabetes Research Foundation. W. Qi is supported by a National Health Medical Research Council Scholarship.
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作者单位：1 Department of Medicine, Kolling Institute, University of Sydney, Royal North Shore Hospital, Sydney; 2 Department of Medicine, University of Sydney, and Royal Prince Alfred Hospital, Sydney; 3 Department of Medicine, St. Vincent?s Hospital, Melbourne, Australia; and 4 Department of Medicine, Unive