作者：Patricia Juárez, M. Magdalena Vilchis-Landeros, José Ponce-Coria, Valentín Mendoza, Rogelio Hernández-Pando, Norma A. Bobadilla, and Fernando López-Casillas作者单位：1 Departamento de Biología Celular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 2 Departamento de Patología, Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán,“ and 3 Unidad de Fisiol $ E2 r5 d; ~8 j ; z& f) o2 w, c& i6 s2 m' J
' @- e# v5 w( t! D
! e) t2 |9 _7 ~5 } 7 E A6 t# H. z) e* P7 a) d5 t7 U$ R
! w: c: x5 y# q5 E% I ! [, e* V/ k. D ) {( p: b+ W6 v' C- o # d3 P' ^, S J
【摘要】8 w6 b7 f) v9 r5 V
Transforming growth factor- (TGF- ) is a key mediator in the pathogenesis of renal diseases. Betaglycan, also known as the type III TGF- receptor, regulates TGF- action by modulating its access to the type I and II receptors. Betaglycan potentiates TGF-; however, soluble betaglycan, which is produced by the shedding of the membrane-bound receptor, is a potent antagonist of TGF-. In the present work, we have used a recombinant form of soluble betaglycan (SBG) to prevent renal damage in genetically obese and diabetic db/db mice. Eight-wk-old db/db or nondiabetic ( db/m ) mice were injected intraperitoneally with 50 µg of SBG or vehicle alone three times a wk for 8 wk. The db/db mice that received vehicle presented albuminuria and increased serum creatinine, as well as glomerular mesangial matrix expansion. The db/db mice treated with SBG exhibited a reduction in serum creatinine, albuminuria, and structural renal damage. These effects were associated with lower kidney levels of mRNAs encoding TGF- 1, TGF- 2, TGF- 3, collagen IV, collagen I, fibronectin, and serum glucocorticoid kinase as well as a reduction in the immunostaining of collagen IV and fibronectin. Our data indicate that SBG is a renoprotective agent that neutralized TGF- actions in this model of nephropathy. Because SBG has a high affinity for all TGF- isoforms, in particular TGF- 2, it is found naturally in serum and tissues and its shedding may be regulated. We believe that SBG shall prove convenient for long-term treatment of kidney diseases and other pathologies in which TGF- plays a pathophysiological role. : A2 |# X2 t% o5 B+ W: ~8 T, t/ q* D 【关键词】 diabetic nephropathy transforming growth factor serum glucocorticoid kinase fibronectin collagen IV mesangial matrix expansion, y9 Q' J/ P! t/ A% X
TRANSFORMING GROWTH FACTOR - (TGF- ) is a multifunctional cytokine involved in the control of cellular proliferation and differentiation, development, immune responses, and tissue repair ( 16, 22 ). Deregulated TGF- actions underlie the pathogenesis of oncogenic, autoimmune, and fibrotic diseases ( 5, 23 ). Among the best-characterized examples of how this deregulation results in disease are the nephropathies. An overwhelming amount of evidence indicates that, independently of the etiology, persistent overproduction of TGF- is an important mediator of kidney disease. It has been shown that overexpression of TGF- in an injured glomerulus induces a "profibrotic" feedback loop that overproduces, among others, extracellular matrix components and TGF- itself, resulting in the onset of disease ( 28, 33 ). The fact that many of these events can be blocked by the administration of TGF- -neutralizing agents not only confirms the relevant pathophysiological role of TGF- but also supports the anti-TGF- strategies as rational treatments for kidney disease ( 6, 28 ). Relevant for the present work is the demonstration that TGF- also plays a role in the nephropathy exhibited by db/db mice ( 14, 36 ). : Z7 Q9 S5 h! U' N* P' u( @! h" m& ]/ h& r, o
TGF- signals through two related transmembrane Ser/Thr kinase receptors, type I and II receptors, which, on heteromerization by the ligand, phosphorylate members of the Smad family of transcriptional regulators that mediate cellular TGF- responses. In addition to type I and II receptors, there are two TGF- coreceptors, betaglycan and endoglin, that control its access to the kinase receptors ( 2, 23 ). Betaglycan, also known as the type III TGF- receptor, is a membrane proteoglycan with a core protein that binds all TGF- isoforms with high affinity. Betaglycan is a positive regulator of TGF- because it increases the affinity of TGF- binding to the type II receptor, enhancing cell responsiveness to TGF- ( 21, 25 ). Although betaglycan was considered for a long time an "accessory" receptor of TGF-, the fact that the betaglycan knockout mouse exhibits an embryonic lethal phenotype indicates otherwise ( 29 ). Adding to the functional complexity of betaglycan is its function as the inhibin coreceptor that mediates its activin antagonism ( 34 ). ' v& U5 ]. {% u( X8 _& Q: K# J# }2 ~* q7 X+ H, }, G
Membrane betaglycan also serves as the precursor for a soluble form of the receptor. Soluble betaglycan (SBG) is shed from the membrane-bound receptor by constitutive and regulated proteolytic cleavages of its extracellular region ( 1, 31 ). In contrast to membrane betaglycan, SBG inhibits TGF- binding to kinase receptors and thereby works as a TGF- antagonist ( 20, 32 ). In previous work, we have employed this property of SBG for the reduction of tumor progression and metastasis in xenograft models of breast and prostate cancer as well as for the treatment of murine tuberculosis, diseases mediated, in part, by TGF- ( 3, 4, 13 ). In the present study, we have taken advantage of the potent TGF- -neutralizing activity of SBG for treating the db/db mouse, a genetic model of type 2 diabetes that develops a nephropathy that resembles the disease in humans ( 7, 27 ). We found that administration of recombinant SBG decreases both structural and functional renal injury in the db/db mouse. Because of its unique biological features, our findings position SBG as a strong therapeutic anti-TGF- agent. 6 [/ f1 {' Q5 }$ B$ ]* ]" R ) E6 S! ?: ^2 k3 Z( w. |/ ?MATERIALS AND METHODS9 N; Z+ e6 [' ?3 S( \! d
# X; u# c& |; j2 dAnimals and experimental design. Male db/db mice (C57BLKS/J) were purchased from Jackson ImmunoResearch Laboratories (Bar Harbor, ME). The db/db mouse, lacking the hypothalamic leptin receptor, is a model of type 2 diabetes mellitus that exhibits hyperglycemia, hyperinsulinemia, and hyperleptinemia associated with hyperphagia and obesity manifesting at 4-7 wk after birth ( 7, 27 ). SBG was prepared as a baculoviral recombinant secreted protein and was purified as described before ( 32 ). SBG or vehicle (PBS) was injected intraperitoneally (ip) three times per week over the 8-wk duration of the experiment. Diabetic male db/db mice and their nondiabetic db/m littermates were randomly divided into four groups as follows: db/m PBS ( n = 8) or db/db PBS ( n = 14), injected with 100 µl PBS as control untreated groups, and db /SBG ( n = 8) or db/db SBG ( n = 14), treated with 50 µg SBG as the treated groups. The SBG dose was selected on the basis of unpublished experiments with rats subjected to Anti-Thy-1 nephropathy (Vilchis-Landeros MM, Juárez P, Mendoza V, Bobadilla NA, Aguilar-León D, Hernández-Pando R, and López-Casillas F, unpublished observations), which showed that an effective therapeutic ip dose for rodents was in the range of 0.8-2 mg/kg. Treatments started at 8 wk of age because 100% of db/db mice become frankly hyperglycemic at this age ( 27 ). All procedures followed were in accordance with our institutional guidelines for animal care. Before death (at 16 wk of age), individual mice were placed in metabolic cages to obtain 24-h urine collections. No sugar was added to the water provided to the mice during the collection period. Urine losses due to evaporation were prevented by overlaying mineral oil in the collection tubes. Serum and urine creatinine were measured with an autoanalyzer (Technicon RA-1000, Bayer, Tarrytown, NY). Creatinine clearance (C cr ) was calculated by the standard formula C cr = UV/P, where U is the concentration of creatinine in urine, V is the urine flow rate, and P is plasma creatinine concentration. 3 r5 H. L( z* o3 ^- X. m* s7 |) Z, [* K7 g! R
Analysis of SBG tissular distribution. SBG was iodinated by the chloramine T method ( 8 ). Groups of two male db/db or db/m (7- to 9-wk-old) mice were injected with 50 µg of cold SBG added to 577,700 counts/min of 125 I-labeled SBG as a tracer in a final volume of 0.1 ml. For death at 0, 0.5, 1, 2, 4, 8, 12, or 24 h, animals were anesthetized by ether inspiration, serum was obtained, and organs were dissected and weighed. Radioactivity in samples was quantified using a Cobra II gamma counter./ v+ r/ K0 {6 O- _1 n. w; F u
* c* ^6 g9 N1 r( W( m1 Q2 m* HUrine albumin assay. Albumin concentrations in 24-h urine samples were measured with a competitive ELISA ( 10 ) in which mouse albumin in the soluble phase competes with albumin immobilized onto microtiter wells (250 ng/well) for binding to a horseradish peroxidase-conjugated anti-albumin antibody (Exocell, Philadelphia, PA). To avoid errors from an incomplete urine collection, albumin excretion was also normalized to urine creatinine. , |, |# q( O$ [3 o" J% A) l9 P; i. v% m$ J6 O f( E% X
Glomerular histology, morphometry, and immunohistochemistry. After death, kidneys were carefully dissected and weighed. All histological studies were done with nonperfused kidneys. It has been reported that the relative degree of glomerular hypertrophy remains the same, with or without perfusion ( 27 ). One section of renal cortex per mouse was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned (5 µm), and stained with periodic acid-Schiff reagent (PAS). Twenty glomeruli were randomly selected from each animal, and the extent of extracellular mesangial matrix was identified by PAS-positive material and measured using a computer-assisted color image analyzer (QWin-Leica, Milton Keynes, Cambridge, UK). The area of the PAS-positive material in the mesangium was factored by the glomerular tuft area to obtain the fraction of mesangial matrix. For immunohistochemistry, deparaffinized tissue sections were rehydrated and blocked with 3% H 2 O 2 in methanol followed by antigen retrieval in a microwave oven in 10 mM citrate buffer, pH 6.0, for 5 min. To reduce background components, tissue sections were treated with antibody diluent (Dako, Carpinteria, CA) diluted 1:100 in PBS and then incubated at 4°C overnight with specific primary antibody. Antibodies against collagen IV and fibronectin were purchased from Abcam (Cambridge, UK). Incubation with secondary biotinylated anti-immunoglobulin followed by horseradish peroxidase-conjugated streptavidin (BioGenex, San Ramon, CA) was done according to the manufacturer?s instructions. Peroxidase activity was revealed with 3-amino-9-ethyl-carbazole (BioGenex) in acetate buffer containing 0.05% H 2 O 2. The sections were counterstained with hematoxylin. For the negative nonimmune control slides, the primary antibody was replaced by nonimmune serum. 4 D' ~6 ~1 y6 c. H1 A/ k3 P: b; o |, D9 d% M# D. w8 X3 u
RT and real-time PCR amplification. Reverse transcription (RT) was carried out using 10 µg of total RNA from the kidney of each mouse. RT was performed at 42°C for 50 min in a total volume of 20 µl using 200 U of Moloney murine leukemia virus Reverse Transcriptase (Invitrogen), 100 pmol of random hexamers (Invitrogen), 0.5 mmol/l of each dNTP (Sigma, St. Louis MO), and 1 x RT buffer (Reaction, Invitrogen). Quantitative real-time PCR amplification was performed using the ABI Sequence Detection System (ABI PRISM 7700). PCR was performed using a Quantitect SYBR Green PCR Kit (Qiagen) in a 25-µl reaction volume containing 1 µl of cDNA and 20 pm of specific 5'- and 3'-primers. A dynamic range was built with each product of PCR on copy number serial dilutions of 1 x 10 8, 1 x 10 7, 1 x 10 6, 1 x 10 5, 1 x 10 4, 1 x 10 3, and 1 x 10 2; all PCRs were performed in triplicate. A total of 40 amplification cycles was performed, an initial incubation step of 15 min at 95°C to activate the enzyme, denaturing at 95°C for 30 s, annealing at 60°C for 30 s, and extending at 72°C for 45 s. Standard curves were calculated referring the threshold cycle (the PCR cycle at which a specific fluorescence becomes detectable) to the log of each cDNA dilution step. Results are expressed as the number of copies of target mRNA normalized to 1/100 of the number of copies of acidic ribosomal protein mRNA. All primers were designed using ABI PRISM 7500 system software and were obtained from Sigma-Aldrich Quimica. Nucleotide sequences of primers used are shown in Table 1.4 p" u! o: E5 ]: ]/ _
# k8 p6 v a/ h8 s
Table 1. Primer sequences used to analyze mRNA levels by real-time RT-PCR9 z$ }( z' ?9 J3 O2 ?; w1 `
9 l$ U0 @3 W& R5 H" ?
Statistical analysis. Data are presented as means ± SE, with n representing the number of animals. Groups were analyzed by one-way ANOVA, and the Newman-Keuls test was used to compare difference among the groups. P 4 c# l' N: L8 P+ T! _( X ; S3 D% A% i. [/ O( wRESULTS 7 H1 k+ J. F7 R0 ~* f 9 S( k6 i7 S( L% h+ ~# g( GPhysiological parameters. To evaluate whether SBG can prevent TGF- -induced kidney damage, we employed genetically obese and diabetic, leptin receptor mutant db/db mice. In the C57BLKS/J genetic background, the db/db mouse exhibits many features of human diabetic nephropathy. Although the db/db mouse does not develop a full-fledged renal insufficiency syndrome ( 7 ), it is a good model for human diabetic nephropathy because it presents albuminuria, increased serum creatinine, glomerular mesangial expansion, and kidney histological lesions resembling those found in the human disease ( 27 ). From 8 to 16 wk of age, diabetic db/db mice and their db/m heterozygous nondiabetic littermates were injected intraperitoneally (ip) with 50 µg of SBG three times a week. Untreated groups received only vehicle (PBS). The db/db mice remained hyperglycemic and glycosuric throughout the experimental period, and because of glycosuria, urine volumes were markedly increased in both db/db groups ( Table 2 ). Body weights at the beginning and at the end of the study were significantly greater in db/db mice than in nondiabetic controls. Kidney weights were significantly greater in db/db compared with db/m mice, and SBG treatment did not modify the kidney weight in either db/db or db/m mice ( Table 2 ). ; o4 B; i _# i+ g9 K$ K4 ^; V" G6 R2 E# m: k/ @0 z
Table 2. Physiological parameters in experimental groups 9 t0 o5 }4 ?" a6 C4 q" G 5 x7 d( ^$ _- W+ r4 F% n' v7 O/ ySBG treatment reduced plasma creatinine and albuminuria in db/db mice. At the end of the study, db/db mice treated with PBS developed renal function impairment, as measured by elevated plasma creatinine and urine albumin excretion ( Fig. 1 ). Similar to what other workers have observed ( 9, 36 ), the plasma creatinine concentration was elevated 2.5-fold in untreated db/db compared with db/m mice. SBG induced a significant reduction in serum creatinine in db/db mice ( Fig. 1 A ), and creatinine clearance was also improved; however, the difference did not reach statistical significance ( Fig. 1 B ). To minimize any error due to incomplete urine collection, the absolute urinary albumin excretion ( Fig. 1 C ) and albuminuria calculated as relative ratio to creatinine excretion ( Fig. 1 D ) were evaluated. Albumin excretion was increased more than fourfold in the db/db PBS group compared with the db/m PBS group. SBG treatment of db/db mice resulted in at least a 50% reduction in albuminuria. SBG did not modify albumin excretion in nondiabetic db/m mice. * H: w. Z' S1 ^; `* f8 U9 j' | U% t4 y8 H) f" L% ], `2 B
Fig. 1. Soluble betaglycan (SBG) treatment of diabetic ( db/db ) mice decreases plasma creatinine and albuminuria. Plasma creatinine ( A ) and creatinine clearance ( B ) were determined in normal nondiabetic ( db/m ) and db/db mice. Gray bars, db/m PBS and db/m BGS mice; black bar, db/db PBS group; open bar, db/db BGS mice. ** P / D% z+ v$ Q. p* U& r
9 k( f% n9 B4 L4 ^
SBG prevented mesangial expansion in db/db mice. Figure 2 A shows representative digitalized photomicrographs of a glomerulus from each experimental group showing diffuse mesangial expansion characterized by an increase in the accumulation of PAS-positive matrix. Figure 2 B depicts a morphometric analysis indicating that in db/db mice the mesangial matrix fraction was expanded 2.4-fold compared with db/m mice. This observation is consistent with previous reports by other workers ( 9, 10, 17, 36 ). In db/db mice, SBG administration prevented mesangial expansion to nearly the values seen in db/m mice. Although SBG treatment significantly reduced the mesangial expansion in db/db mice, it did not affect mesangial area in db/m mice. As reported by other workers that have characterized kidney pathology in the db/db mouse ( 27, 36 ), none of our experimental groups presented significant pathological changes in the tubulointerstitial compartment (data not shown).8 r0 f) y) \9 D/ g& D8 d
) L$ I( B2 x2 k" X N
Fig. 2. SBG treatment prevents mesangial matrix expansion in db/db mice. A : periodic acid-Schiff (PAS)-stained kidney sections from normal db/m and diabetic db/db mice treated with PBS or SBG. B : quantitative measurement of extracellular mesangial matrix expansion. Mesangial matrix fraction was calculated as the ratio of mesangial area to glomerular area. The average value was obtained from analyses of 20 glomeruli/mouse. ** P ; B& s6 u2 i, F) E, G4 M8 C' @& P8 p! r
Body distribution of SBG. To determine SBG body distribution in db/db mice, 50 µg of SBG along with a tracer amount of 125 I-labeled SBG were injected ip into 7- to 9-wk-old db/db or db/m mice and its distribution was determined at diverse time intervals by measuring the amount of 125 I in blood and several tissues such as the kidney, liver, lungs, and heart. As shown in Fig. 3 A, SBG quickly entered the blood circulation, reaching a peak at 30 min after injection and slowly decreasing to become virtually undetectable at 24 h. Similar half-lives of circulating 125 I-labeled SBG, of 4 h, were observed in both diabetic and nondiabetic mice. Importantly, 125 I-labeled SBG reached the kidneys in diabetic and nondiabetic mice, concentrating 10% of the label after 30 min of injection ( Fig. 3, B and C ). 125 I-labeled SBG was detected in other organs, even at greater amounts than in kidneys as in the case of liver, which accumulated up to 22% of the label at 30 min postinjection. However, considering the absolute organ weight, the kidneys are the organs that better concentrated 125 I-labeled SBG, with values that are two to three times larger than for the liver. After the 30-min maximum, 125 I-labeled SBG disappeared from kidneys, liver, lungs, and heart, following kinetics that mirror its serum levels. Although the graphs shown in Fig. 3 may suggest that 125 I-labeled SBG clears from the serum and kidneys of db/db mice more quickly than from db/m mice, the limited amount of data forbids any statistically significant conclusion. On the other hand, since our SBG treatment schedule consisted of three injections every week and considering that the kinetics exhibited by 125 I-labeled SBG, it is safe to conclude that there is at least a 24-h period of a very low level of SBG in the blood and kidney between every injection. Nonetheless, as will be shown by data in the following sections, despite its discontinuous serum levels, SBG effectively decreased the renal expression of genes upregulated by TGF-.9 ^ q+ A: b; w% ]% Z
$ D% y/ C `. y2 Q; r: z5 N9 \Fig. 3. SBG serum and tissular distribution. 125 I-SBG was administered to 14 db/db or 14 db/m mice, and at the indicated times 2 mice/group were killed to determine its distribution in serum ( A ) and several tissues ( B and C ) as described in MATERIALS AND METHODS. 125 I-SBG [577,700 counts/min (cpm)] was injected into each mouse. The amount of 125 I-SBG in serum was expressed as cpm/m, while in kidneys (gray bars), liver (open bars), lungs (hatched bars), and heart (black bars) as the percentage of the injected label detected in the organ. Kidneys show the greatest accumulation of 125 I-SBG in the analyzed organs when calculated as 125 I-SBG/g tissue (not shown).8 W4 H; Y/ |/ K9 {
! O" h7 K7 c6 _" S7 h/ J% ~
TGF- upregulation in kidneys of db/db mice was reversed by SBG. As part of the TGF- pathophysiological mechanism, it has been previously shown that this profibrotic factor upregulates diverse genes in the kidney of diabetic mice. One of the most relevant is TGF- 1, an effect that has been proposed as responsible for a positive feedback loop that keeps the TGF- signal active during the progression of nephropathies ( 28, 33 ). Therefore, we used real-time RT-PCR to determine the levels of expression of TGF- 1 and the other two mammalian TGF- isoforms in our experimental groups using the primers indicated in Table 1. Figure 4 presents a quantitative analysis of TGF- 1, TGF- 2, and TGF- 3 mRNAs showing increases of 2.8-, 2.0-, and 5.0-fold, respectively, in the kidney of control untreated db/db mice compared with their nondiabetic db/m littermates. Importantly, in the diabetic mice treated with SBG it significantly prevented the upregulation of all three TGF- isoform mRNAs, decreasing their levels similar to those in nondiabetic db/m mice. SBG treatment did not show any significant effect on the levels of the TGF- mRNAs in db/m mice. ' N) ?/ z( ~9 w; j: O. s6 O$ q % L% _& q Z- Y2 p( C9 ^Fig. 4. SBG downregulates the expression of transforming growth factor (TGF)- 1, TGF- 2, and TGF- 3 in db/db mice. The intrarenal level of TGF- 1 ( A ), TGF- 2 ( B ), and TGF- 3 ( C ) mRNAs was determined by quantitative real-time RT-PCR as described in MATERIALS AND METHODS and normalized using the levels of the mRNA for acidic ribosomal protein (ARP), which has been reported as one of the most reliable standards for quantitative real-time PCR experiments ( 11 ). Gray bars, db/m PBS and db/m BGS mice; black bar, db/db PBS group; and open bar, db/db BGS group. ** P # ]* A' i9 z+ A/ p, K/ O 3 C1 Z) c- ~+ k |, Z: W# y- ~SBG decreased fibronectin and collagen IV in kidneys of diabetic mice. To test whether the downregulation of the three TGF- isoforms had any effect on extracellular matrix components, we evaluated by immunohistochemistry glomerular fibronectin and collagen IV. These proteins are well-characterized markers that are increased by TGF- in diverse models of nephropathy ( 9, 28 ). Compared with their nondiabetic littermates, the untreated db/db mice had a threefold increase in glomerular staining for fibronectin and collagen IV ( Fig. 5 ). Treatment with SBG significantly decreased the levels of these proteins in db/db mice without any effect on db/m mice.( S) V, g6 [+ k
) J2 U# m8 u4 g7 mFig. 5. SBG decreases the levels of fibronectin and collagen IV in db/db mice. Glomeruli in kidneys from SBG-treated and untreated (PBS) db/db or db/m mice were immunostained with antibodies specific for fibronectin ( A ) or collagen IV ( B ). Morphometric quantification of the fibronectin or collagen IV staining in our 4 experimental groups is shown in C and D, respectively.& ~' k& z2 B7 t5 A" P
% M5 P8 x, B8 I1 U P, i8 L. s: ~
SBG downregulated TGF- -responsive genes in kidneys of diabetic mice. Figure 6 depicts the expression of other TGF- gene targets evaluated by real-time RT-PCR using the primers shown in Table 2. The levels of fibronectin, collagen IV, collagen I, and serum glucocorticoid kinase (SGK) mRNAs in the kidneys of db/m mice were barely detectable by this technique. As expected, the untreated db/db mice exhibited large amounts of these mRNAs. In contrast and in accordance with our immunostaining results, SBG administration to db/db mice resulted in a significant reduction in collagen I, collagen IV, fibronectin, and SGK mRNAs by 3-, 3.3-, 2- and 2-fold, respectively. Similar reductions in the expression of the "ECM markers" collagens and fibronectin by means of TGF- inhibition have been reported before ( 9, 28 ). However, to our knowledge this is the first time that SGK, a "functional marker" of kidney disease ( 18 ), is shown to be downregulated by a TGF- inhibitor. SGK is a serine-threonine kinase that is upregulated by glucose and TGF- and is involved in the regulation of sodium transport; thus it has been proposed as a pathophysiological mediator of diabetic nephropathy ( 18 ). Interestingly, although the treatment with SBG did not completely abolish the expression of SGK, it was capable of decreasing it to 60% despite the continuous hyperglycemia exhibited by these mice ( Fig. 6 D ). / v2 _2 s3 P# B) u7 S1 Y. V' l9 z0 I N ^7 z E3 E
Fig. 6. Effect of SBG on the expression of TGF- -responsive genes in kidneys from db/m and db/db mice. The levels of the indicated mRNAs were determined by quantitative real-time PCR and normalized using the levels of ARP mRNA for collagen IV ( A ), collagen I ( B ), fibronectin ( C ), and SGK ( D ). Gray bars, db/m PBS and db/m BGS mice; black bar, db/db PBS group; open bar, db/db BGS. ** P , p, N6 k: a2 ?. e
4 u# H3 U% J/ D$ \
DISCUSSION 5 |4 G/ y8 i8 d% Y3 A4 j( } 9 z. ]# y( a6 x! v7 hThe role of TGF- as the key mediator of kidney disease has been extensively documented and has been reviewed elsewhere ( 28, 33 ). Independently of the etiology, TGF- plays this central pathophysiological role in experimental and human nephropathies. The kidney damage observed in the genetically obese and diabetic db/db mouse is also associated with an increase in renal TGF- signaling ( 14, 36 ). Therefore, we employed the db/db mouse to determine whether SBG, a potent inhibitor of TGF- ( 32 ), could block TGF- in vivo and thereby ameliorate the progression of renal injury. 0 M [! d" y& {4 |0 y $ T8 X& T. ^8 o; X3 ]- mThe administration of SBG to db/db mice from 8 to 16 wk of age, a period during which overt nephropathy progresses, resulted in a reduction in kidney damage. Plasma creatinine concentration, albuminuria, and mesangial matrix expansion, which are increased in db/db mice at week 16 ( 9, 10, 17, 27, 36 ), were significantly reduced by the treatment with SBG ( Figs. 1 and 2 ). These findings show that SBG treatment effectively ameliorated key structural and functional aspects of renal injury in db/db mice despite the hyperglycemic state of these animals. One relevant finding of the present study is that despite its short circulating half-life, SBG administration had a lasting effect on the expression of well-known TGF- gene targets. We evaluated SBG tissue distribution after ip administration and found that it becomes readily detectable in serum and diverse organs, especially the kidneys, after 30 min of injection ( Fig. 3 ). SBG remains in blood circulation with a half-life of 4 h in db/db and db/m mice, becoming barely detectable in blood and tissue after 24 h ( Fig. 3 ). Importantly, despite not exhibiting constantly high serum concentrations, SBG was able to downregulate TGF- -responsive genes that are usually overexpressed in the kidneys of diabetic animals; one such gene is the same TGF-. The data in Fig. 4 corroborate the previously known renal overexpression of TGF- 1 in db/db mice and also document that the other two TGF- isoforms are also upregulated in the kidneys of these animals. As expected, SBG significantly reduced the expression of all TGF- isoforms. This finding is relevant because, despite the fact that TGF- 1 is the best-studied isoform in regard to renal diseases, there is evidence suggesting similar pathophysiological roles for the other two isoforms. For instance, it has been shown that TGF- 2 and TGF- 3 also have fibrogenic effects on cultured kidney cells ( 35 ) and in vivo the injection of recombinant human TGF- 2 to rats and mice induces renal fibrosis ( 15, 19 ). In addition to its effect on the expression of the TGF- isoforms, SBG also reduced the kidney levels of collagen IV and fibronectin, as evidenced by a decrease in their immunostaining in SBG-treated db/db kidneys ( Fig. 5 ). SBG also reduced the overexpression of renal collagen I, collagen IV, fibronectin, and SGK mRNAs in the db/db mouse ( Fig. 6 ). SGK is upregulated by TGF- in diabetic kidneys, and recent studies have indicated that it may have a prominent role as a "functional mediator" of TGF- -dependent kidney damage ( 18 ). To our knowledge, this is the first report of SGK mRNA upregulation in db/db mice, as well as its reduction by a TGF- inhibitor. ! l T! U! G- y" f! O " W3 G8 r9 L) _5 U3 ZGiven its powerful in vitro and in vivo anti-TGF- effects ( 3, 4, 13, 20, 32 ), it is very likely that SBG renoprotective actions in the db/db mouse arise from TGF- neutralization. In support of this, other anti-TGF- agents have been tested in the db/db model and shown to restore functional and structural markers of kidney disease. For instance, Ziyadeh et al. ( 36 ) used a monoclonal pan-specific anti-TGF- antibody (2G7) that significantly reduced mesangial expansion, plasma creatinine, and creatinine clearance, but failed to reduce albuminuria. In another study, Sugaru et al. ( 30 ) demonstrated that SMP-534, an agent that apparently inhibits TGF- via inhibition of p38 MAPK, reduced mesangial expansion, albuminuria, and creatinine clearance, but failed to reduce serum creatinine. Overall, SBG, 2G7, and SMP-534 improved kidney function in db/db mice; however, it is difficult to account for the peculiarities of their therapeutic responses, especially when they are regarded as anti-TGF- agents and therefore should produce equivalent effects. One explanation may reside in their chemical nature and distinct mechanism of action. This is relevant because it is has been proposed that the cross talk between the Smad and other signaling pathways may be important for the onset of TGF- -mediated kidney damage ( 12, 17, 33 ). Another explanation may be found in the complexity of the pathophysiology of diabetic kidney damage. Examples are the differential effects of these anti-TGF- agents on albuminuria. As recently reviewed by Shankland ( 26 ), proteinuria is a complex problem whose origin involves several factors (VEGF, nephrin phosphorylation, etc.). The question of which of these factors or pathogenic mechanisms is selectively targeted by SBG therapy is still unanswered and remains the subject of our investigations. 7 j9 D9 }# j3 B X! O 8 n, J9 a4 c7 D6 A" pIn summary, we have shown that SBG treatment of db/db mice significantly ameliorates the progression of kidney dysfunction, without any apparent negative side effect, as indicated by the lack of gross macroscopic damage of internal organs. This is in agreement with a study that revealed only minimal immunological effects in normal mice after a 12-wk-long treatment with an anti-TGF- antibody ( 24 ). Nonetheless, it is clear that a comprehensive study of possible complications derived from long-term systemic inhibition of TGF- is still due. Finally, from all the diverse anti-TGF- agents that have been tested in experimental therapeutics, SBG has some biological properties that could turn advantageous for long-term treatment. For example, since SBG is normally found in serum and extracellular fluids, it is unlikely that its long-term systemic administration would elicit an immune response. Regarding its TGF- -neutralizing potency, SBG and anti-TGF- antibodies have similar potencies against the TGF- 1 isoform; however, because of its higher affinity for TGF- 2, SBG is 10 times more potent against this isoform ( 32 ). If TGF- 2 turns out to be as relevant as TGF- 1 to kidney disease, a pending issue that deserves comprehensive study, then SBG?s TGF- 2 isoform selectivity would be a tremendous asset. However, the property of SBG that makes it stand out from the other inhibitors is the fact that its in vivo generation is amenable to regulation. Ever since the discovery that SBG was, in opposition to its membrane form, an inhibitor of TGF- ( 20 ), it was proposed that this receptor could play a relevant role in the physiological regulation of TGF-, working like a "switch" that could turn on its functions as a membrane-bound receptor or turn them off as a soluble one ( Fig. 7 ). With the discovery that the shedding of membrane betaglycan is a regulated process ( 31 ), and given its widespread expression in almost every cell lineage, it is not difficult to foresee new pharmacological strategies for an anti-TGF- therapy based on betaglycan shedding. Namely, molecules that could promote its shedding would become effective anti-TGF- agents. Further work is in progress in our laboratory to explore the feasibility of these novel strategies.) ^9 ?1 p7 v! ^0 Z4 T
$ S7 n9 a* s3 D# E# Z7 t6 a
Fig. 7. Betaglycan (BG) dual modulation of TGF- activities. BG, also known as the type III TGF- receptor, potentiates TGF- actions when it is membrane bound. Shedding of BG extracellular region generates the soluble form of the receptor. Soluble BG still binds TGF- with the high affinity of the membrane BG, but instead of "presenting" it to the type II receptor, soluble BG "sequesters" it and therefore neutralizes its actions ( 20, 32 ). Unique among other TGF- inhibitors is the fact that BG may be subject to regulated shedding of its ectodomain ( 31 ), making possible, in principle, control of the relative ratio of the membrane and soluble forms of the receptor, providing a way to switch TGF- actions on or off.+ l6 f! h! {* X. s: n7 D
8 T6 N' R$ E V3 z) Z% TGRANTS9 `, r7 s2 l1 G* b
; X I- l: r0 u
This work was supported in part by an International Research Scholar Grant from the Howard Hughes Medical Institute and by Grants 37749N and MO316 from the Consejo Nacional de Ciencia y Tecnología (to F. López-Casillas).+ | \( @0 @& e9 `3 O0 m% m% ~
, t5 A" F) d, r0 B# y
ACKNOWLEDGMENTS: ]7 G0 Z. E! l6 j* ]" D6 y
5 H' `3 X# s. N* ^* eWe thank María de los Remedios Rámirez Rángel for assistance with immunohistochemical techniques, Erika L. Monterrubio Flores and Guadalupe Hiriart Valencia for assistance in glomerular histology, Jazmín M. Pérez-Rojas and Joyce Trujillo for assistance with renal function tests, Diana Aguilar-León for assistance with real-time PCRs, and Drs. Claudia Rivera and Héctor Malagón for assistance with the care of db/db mice.8 E* g- P8 t; {# j" z3 K
【参考文献】 & ?* [+ i8 L+ E4 G- p1 ~ Arribas J, López-Casillas F, Massagué J. Role of the juxtamembrane domains of the transforming growth factor- precursor and the -amyloid precursor protein in regulated ectodomain shedding. J Biol Chem 272: 17160-17165, 1997.8 w6 |8 k4 {2 n( `
; Y$ ^% _6 y# j! O0 U
) ` @* H2 v' \) R! ~, `1 n
5 [+ f% E1 R7 OAttisano L, Wrana JL, López-Casillas F, Massagué J. TGF- receptors and actions. Biochim Biophys Acta 1222: 71-80, 1994.* A7 Q: g: ~# F' Z/ i) @* P) v
4 f6 V# Z; q$ t/ j" M
2 [! r# S/ u8 P; o) m
6 j: a% y, P2 j8 y9 y& n2 S
Bandyopadhyay A, López-Casillas F, Malik SN, Montiel JL, Mendoza V, Yang J, Sun LZ. Antitumor activity of a recombinant soluble betaglycan in human breast cancer xenograft. Cancer Res 62: 4690-4695, 2002.5 M4 \& s, E) D4 ]6 l
) {2 D2 n! ^% K* J- _
5 K6 ^4 x# a% o+ x- t# P4 X' U5 r" A, i, o- P3 G/ n
Bandyopadhyay A, Wang L, López-Casillas F, Mendoza V, Yeh IT, Sun LZ. Systemic administration of a soluble betaglycan suppresses tumor growth, angiogenesis, and matrix metalloproteinase-9 expression in a human xenograft model of prostate cancer. Prostate 63: 81-90, 2005.$ u8 @7 Z7 q8 j* b+ r
) t& {0 e2 L& R3 t
# E& O2 b2 ]3 y. }+ m
- {9 w- A& I; {1 g$ x; i& c7 b! N
Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor in human disease. New Engl J Med 342: 1350-1358, 2000.; |0 N4 h# c5 |2 O. |
& W# N: B! J5 i7 p! L Q( s / ]$ U! I& O" f5 p0 N& z7 @# x+ Y # J: G# M- ~; m- VBorder WA, Okuda S, Languino LR, Sporn MB, Ruoslathi E. Suppression of experimental glomerulonephritis by antiserum against transforming growth factor. Nature 346: 371-374, 1990. c* B1 h. u0 m) [0 ]& t
% ]$ W% Y. X$ w! N$ F4 L : U, {" a# x j ) ?% @& y6 K2 R6 Q) `7 V) v9 cBreyer MD, Böttinger E, Brosius FC III, Coffman TM, Harris RC, Heilig CW, Sharma K. Mouse models of diabetic nephropathy. J Am Soc Nephrol 16: 27-45, 2005.: l; N4 d$ J" Q$ {! `- r
/ \* ?5 t6 C4 Z
. u) N0 D! g! w* s% V4 A: j9 d4 c2 k
; K" @9 C0 G- y* a- V; R% @Cheifetz S, Hernandez H, Laiho M, Dijke P, Iwata KK, Massagué J. Distinct transforming growth factor- (TGF- ) receptor subsets as determinants of cellular responsiveness to three TGF- isoforms. J Biol Chem 265: 20533-20538, 1990.9 m, ^9 h* M- X! C/ ~
# n" }1 a9 n9 Y- @+ W
. a0 I8 b. v$ w
. T/ i5 P! @$ I" A; y t1 mCohen MP, Lautenslager GT, Shearman CW. Increased urinary type IV collagen marks the development of glomerular pathology in diabetic db/db mice. Metabolism 50: 1435-1440, 2001. 6 V- r! D4 g3 s7 `$ s! [& b# M9 c- B- }
1 j! Z {. {1 ^8 e( B4 M
' Q* H0 B5 e/ s" [1 g& r
Cohen MP, Sharma K, Jin Y, Hud E, Wu VY, Tomaszewski J, Ziyadeh FN. Prevention of diabetic nephropathy in db/db mice with glycanated albumin antagonists: a novel treatment strategy. J Clin Invest 95: 2338-2345, 1995.$ I% d3 Y$ d9 G7 S/ x
) r+ P1 N0 o% y, h7 U 7 H. \2 b% n' d2 Z& N $ s5 W1 Z+ p9 o' v% i0 j* Q8 a& L/ z. KDheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37: 112-118, 2004./ f8 L# ^, J' m- J V }2 C/ W' g
3 g2 }* D: S1 w8 Z/ ^ 4 e( Q/ ^6 x B$ C8 p. i" }& N; ]9 ~# \# c6 c
Feliers D, Duraisamy S, Faulkner JL, Duch J, Lee AV, Abboud HE, Choudhury GG, Kasinath BS. Activation of renal signaling pathways in db/db mice with type 2 diabetes. Kidney Int 60: 495-504, 2001.' J: z( D: \; L. ?( O+ t' d
4 b( U+ ]2 X B7 S
, h( \+ Z" I9 C& J) e4 A' f
V! l% M! I' N6 s+ K/ d b$ q5 P
Hernandez-Pando R, Orozco-Esteves H, Maldonado HA, Aguilar-Leon D, Vilchis-Landeros MM, Mata-Espinosa DA, Mendoza V, López-Casillas F. A combination of a transforming growth factor- antagonist and an inhibitor of cyclooxygenase is an effective treatment for murine pulmonary tuberculosis. Clin Exp Immunol 144: 264-272, 2006. ( C, C0 t+ x3 t% L- z) Y" L! c; u* ?" f
! b2 ?: ~& S$ V; v
- f w; G, t" T) R
Hong SW, Isono M, Chen S, Iglesias-de la Cruz MC, Han DC, Ziyadeh FN. Increased glomerular and tubular expression of transforming growth factor- 1, its type II receptor, and activation of the Smad signaling pathway in the db/db mouse. Am J Pathol 158: 1653-1663, 2001.' _5 [3 i M, w
9 Y4 g4 v9 D W- e $ g8 j3 Q6 W5 V* Z* J7 m3 ]7 ]- p/ K" N j# v8 a
Kelly FJ, Anderson S, Thompson MM, Oyama TT, Kennefick TM, Coreless CL, Roman RJ, Kurtzberg L, Pratt BM, Ledbetter SR. Acute and chronic renal effects of recombinant human TGF- 2 in the rat. J Am Soc Nephrol 10: 1264-1273, 1999. # \1 X g5 M, \ P; ]* ]5 n& p" y1 K% N. }
7 z! x- v1 Y. K+ T8 I5 g, ~1 ?! n& G2 y) R
Kingsley DM. The TGF- superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 8: 133-146, 1994.% z$ T# i9 W v1 }$ O) A
. x# |' b1 Q1 E+ C9 t" f* U$ F# |
( F; x3 R" n9 `* `3 r0 ~3 y1 R x 2 U. [: B/ y' O; rKoya D, Haneda M, Nakagawa H, Isshiki K, Sato H, Maeda S, Sugimoto T, Yasuda H, Kashiwagi A, Ways DK, King GL, Kikkawa R. Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes. FASEB J 14: 439-447, 2000. 5 n# B3 }0 t( O8 q' `- `* H! T: m2 L0 N/ a9 @. j7 t, ]( A* D
! E7 i. {+ F3 U! X. p) ^7 B8 ?9 [. c9 Z8 V8 S
Lang F, Klingel K, Wagner CA, Stegen C, Wärntges S, Friedich B, Lanzendörfer M, Melzig J, Moschen I, Steuer S, Waldegger S, Sauter M, Paulmichl M, Gerke V, Risler T, Gamba G, Capasso G, Kandolf R, Helbert SC, Massry SC, Bröer S. Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy. Proc Natl Acad Sci USA 97: 8157-8162, 2000.( k- B8 d/ P( C: z2 o
/ k( E" b9 P( S! M4 m5 Y1 G , H$ Q: |" t X/ A3 X' b& Y3 l7 G! C6 x. \& S1 J
Ledbetter S, Kurtzberg L, Doyle S, Pratt BM. Renal fibrosis in mice treated with human recombinant transforming growth factor- 2. Kidney Int 58: 2367-2376, 2000. : Z1 ]. F2 y R8 R" r / P! E% C4 A& }7 r$ F& C$ {: c, ~/ U7 T9 D( }
9 }& H" K- q& I x+ y" E; q
López-Casillas F, Payne HM, Andres JL, Massague J. Betaglycan can act as a dual modulator of TGF- access to signaling receptors: mapping of ligand binding and gag attachment sites. J Cell Biol 124: 557-568, 1994. $ v' y8 J6 e2 H6 g# \8 b; r8 d/ w! ]' R
' {5 }/ z& @0 M, v$ W- I f
! P$ u: I) F# ^/ e& xLópez-Casillas F, Wrana JL, Massague J. Betaglycan presents ligand to the TGF signaling receptor. Cell 73: 1435-1444, 1993. + x; ~5 Q! T+ v9 L 6 m% s. X2 N4 `8 g+ D& p" j0 [3 C2 G0 x& v. q% r! |6 g( v
' a- Q& N- D; y* T; g1 v
Massagué J. The transforming growth factor- family. Annu Rev Cell Biol 6: 597-641, 1990./ @/ C' N A9 z1 {4 m/ k4 C, {
( P# `6 {0 d1 A7 @. J3 Z) {) Y/ v. R1 ?6 e! e0 O
# H; E' K$ D# yMassague J, Blain SW, Lo RS. TGF signaling in growth control, cancer, and heritable disorders. Cell 103: 295-309, 2000.4 \2 C; |2 y) b1 p. {* N+ {
8 P- a2 E8 C* a r# f7 r' v) Y5 x0 z
' b: }+ s2 @4 ~% r( n- r0 Y* }/ D9 y: i$ o' a% j; A: w- s/ W
Ruzek MC, Hawes M, Pratt B, McPherson J, Ledbetter S, Richards SM, Garman RD. Minimal effects on immune parameters following chronic anti-TGF- monoclonal antibody administration to normal mice. Immunopharmacol Immunotoxicol 25: 235-257, 2003." W3 G+ k; M) x( a) B, J
2 c% z4 {* R! F7 R+ f) `$ y+ c2 M s0 k0 R2 v5 F# s
2 \) ~9 E1 f; sSankar S, Mahooti-Brooks N, Centrella M, McCarthy J, Madri JA. Expression of transforming growth factor type III receptor in vascular endothelial cells increases their responsiveness to transforming growth factor 2. J Biol Chem 270: 13567-13572, 1995.- W: o2 I- b1 I+ h. C
1 {: t% [5 l/ A
- }7 ]9 X1 B, L- c' {1 a' W
& c# L7 c4 F% Y9 S, T( V# J" A. JShankland SJ. The podocyte?s response to injury: role in proteinuria and glomerulosclerosis. Kindey Int 69: 2131-2147, 2006. 8 v1 [4 W! x1 o3 H5 c0 u2 v/ { " n, e3 v' r9 S4 e2 j( C 4 f" ?. _) w0 ^) W9 e& \ 0 J* j( v9 Z# v* B; Q, `9 X fSharma K, McCue P, Dunn SR. Diabetic kidney disease in the db/db mouse. Am J Physiol Renal Physiol 284: F1138-F1144, 2003. : f6 B) X* Q7 u+ r3 J5 [% `' j . t$ E. I( {# t) B' @ , g2 A! N1 _* Q9 }% X+ h ]! S- i$ q( h# l a+ {, J
Sharma K, Ziyadeh FN. The emerging role of transforming growth factor- in kidney diseases. Am J Physiol Renal Fluid Electrolyte Physiol 266: F829-F842, 1994.+ _1 \: G* s; A: @) d$ R
+ c! H3 J0 ?7 m& {3 ]' W. j- @) d/ c! w
/ @* F0 N$ u' D) R$ B: I& T
m* U! Q w( V- p$ F( b- p& jStenvers KL, Tursky ML, Harder KW, Kountouri N, Amatayakul-Chantler S, Grail D, Small C, Weinberg RA, Sizeland AM, Zhu H. Heart and liver defects and reduced transforming growth factor 2 sensitivity in transforming growth factor type iii receptor-deficient embryos. Mol Cell Biol 23: 4371-4385, 2003.- k2 C8 m$ \: B1 z- e3 l" Y
/ a U3 ?! p2 w8 H 2 R* c2 M' b6 X( o" {' s3 B% I/ h# v+ M( n1 |
Sugaru E, Nakagawa T, Ono-Kishino M, Nagamine J, Tokunaga T, Kitoh M, Hume WE, Nagata R, Taiji M. SMP-534 ameliorates progression of glomerular fibrosis and urinary albumin in diabetic db/db mice. Am J Physiol Renal Physiol 290: F813-F820, 2006. + M1 e2 }" A! b7 Q, G( b/ ~7 S; o2 L0 Z2 |; P" X* l2 [
+ R2 R2 S9 ~" Y- o/ B5 k
* I+ @3 i; j0 d" lVelasco-Loyden G, Arribas J, López-Casillas F. The shedding of betaglycan is regulated by pervanadate and mediated by membrane type matrix metalloprotease-1. J Biol Chem 279: 7721-7733, 2004.3 S% L8 J3 b) P4 G( C+ U# q* D
6 c$ ?9 ?+ `% `1 | _ ' P( G9 v5 Z4 c : X' N7 N- a8 x8 X' |Vilchis-Landeros MM, Montiel JL, Mendoza V, Mendoza-Hernández G, López-Casillas F. Recombinant soluble betaglycan is a potent and isoform-selective transforming growth factor- neutralizing agent. Biochem J 355: 215-222, 2001. 2 | C# L9 l9 ]/ K0 H) w* X 5 K, | k6 q# v8 s ; L; f1 Z6 { [4 x1 y ) l: F/ ^( Q x1 |, W% n0 L6 sWang W, Koka V, Lan H. Transforming growth factor- and Smad signaling in kidney diseases. Nephrology 10: 48-56, 2005.2 P: r1 H5 S) Z( C0 d$ q) g
5 J0 Z, d; s* m0 g+ B+ J4 G: G: E- |8 H7 O3 o# g$ ]
0 b( a! j/ _7 X0 P
Wiater E, Harrison CA, Lewis KA, Gray PC, Vale WW. Identification of distinct inhibin and transforming growth factor -binding sites on betaglycan. Functional separation of betaglycan co-receptor actions. J Biol Chem 281: 17011-17022, 2006. . C7 V4 a7 K( O8 @* | 6 ~+ P& Z6 z4 |6 @- p S! N: ], Y8 a! o0 N# Z) X4 a. e t8 j: z% R& z; w
Yu L, Border WA, Huang Y, Noble NA. TGF- isoforms in renal fibrogenesis. Kidney Int 64: 844-856, 2003. x/ F; [; V7 I7 k. O3 T ) j: c$ Y8 o( n 0 X! D" w# Q% r8 b : J" N6 D1 a, F3 Z* e9 y1 TZiyadeh FN, Hoffman BB, Han DC, Iglesias-de la Cruz MC, Hong SW, Isono M, Chen S, McGowan TA, Sharma K. Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal anti-transforming growth factor- antibody in db/db diabetic mice. Proc Natl Acad Sci USA 97: 8015-8020, 2000.作者: laoli1999 时间: 2015-6-4 17:41