AT 1 receptor blockade prevents interstitial and glomerular apoptosis but not fi - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 24 下一页

返回列表

查看: 432167\|回复: 233	go AT 1 receptor blockade prevents interstitial and glomerular apoptosis but not fi [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-22 09:37 |只看该作者 |倒序浏览 |打印

作者：Anni Eskild-Jensen,, Lene Fogt Paulsen,, Lise Wogensen,, Ping Olesen,, Lea Pedersen,, Jørgen Frøkiær,,, and Jens Randel Nyengaard,作者单位：1 Department of Clinical Physiology and Nuclear Medicine and 2 Research Laboratory of Biochemical Pathology, Aarhus University Hospital, and 3 Stereology and Electron Microscopy Research Laboratory and MIND Center, 4 The Water and Salt Research Center, and 5 Institute of Clinical Medicine, Universit 9 ^- `$ O( H* W* N! Z) ]

   ! G8 G/ v2 T) f

                     ! O/ ?7 I9 c4 l% b( b
         ) z* v4 |* z: I' q- C( d
         - ]1 R8 b- Q6 c0 _1 v7 ]& V  ]
         - S) }% C: g. h4 n+ ~. O
         8 S7 d8 B. g/ S
                    N. p& r" o, H
      ) z4 B% m1 B6 O$ W

      - K1 I' g% z, o; a
      【摘要】
   Obstruction-induced fibrosis is a leading cause of end-stage renal failure in children. The pathophysiological mechanisms may involve apoptosis and the renin-angiotensin system. We studied apoptosis and fibrosis in a well-established neonatal pig model with unilateral partial ureteral obstruction (PUUO) induced during ongoing nephrogenesis in 2-day-old piglets. The role of angiotensin II (ANG II) was studied using the AT 1 receptor blocker CV-11974 (0.12 mg/h candesartan from age 23 to 30 days). At day 30 the kidneys were perfusion fixed and fibrosis, apoptosis, and tubular lengths were quantitated using stereological methods, picro Sirius red staining, and immunohistochemical techniques identifying activated caspase 3, aquaporin-2 (AQP2), and von Willebrand factor. The collagen content was assessed by hydroxyproline density. Neonatal induced PUUO increased interstitial and glomerular cell apoptosis and fibrosis. At this stage, PUUO did not increase tubular cell apoptosis or decrease tubular length and cell number. AT 1 receptor blockade prevented the PUUO-induced interstitial and glomerular cell apoptosis but did not attenuate fibrosis. In conclusion, AT 1 receptor blockade after the end of nephrogenesis may prevent interstitial and glomerular cell apoptosis but not fibrosis, suggesting that pathways not involving AT 1 receptor stimulation contribute to neonatal obstruction-induced fibrosis or that prevention of interstitial cell apoptosis counteracts a potential antifibrotic effect of AT 1 receptor blockade in this pig model of congenital obstructive nephropathy. Our results demonstrate that ANG II plays a role in PUUO-induced glomerular cell apoptosis. & L% t) u9 z5 ]5 b( t9 \8 k
      【关键词】 angiotensin II caspase neonatal stereology tubular
   CONGENITAL OBSTRUCTION OF the urinary tract is the leading cause of end-stage renal failure in children ( 3 ). A major factor in the loss of renal function is progressive interstitial fibrosis characterized by excess accumulation of extracellular matrix replacing normal kidney structures. The process of fibrosis is initiated by the obstruction and may continue even in the event of obstruction relief, as shown in a neonatal rat model ( 13 ). This indicates that the response of the immature kidney to obstruction may not be abolished by surgical removal of the narrow ureteral segment. Furthermore, it demonstrates the necessity of developing new therapeutic approaches which specifically target cellular fibrotic pathways initiated by the obstruction. Aimed at preventing the progressive loss of normal renal tissue during congenital obstruction in children, it is therefore of great importance to understand the mechanisms of fibrosis formation in the developing kidney. Moreover, nephron loss may be a critical consequence of congenital partial unilateral ureteral obstruction (PUUO), and recently we demonstrated that neonatal obstruction of the multipapillary pig kidney was associated with a significant reduction in the number of glomeruli although long-term renal function was preserved ( 24 ).7 y6 o: _- Q; E5 @5 Z3 l, Z

The pathogenesis of obstruction-induced fibrosis has primarily been studied in vivo in rodent models where important knowledge on cellular pathways and mechanisms has been achieved ( 1, 36 ). However, differences between animal species may prove important when the effect of neonatal obstruction on renal fibrosis is being studied. In a recent study comparing neonatal obstruction in mice and rat kidneys, epidermal growth factor proved to have opposing effects on renal apoptosis in the two species ( 34 ). Such differences may have important implications if growth factors or other modulators of obstruction-induced fibrosis are to be used as therapeutic agents in congenital renal disease in children. Thus, before a move to clinical trials testing the potential of modulating factors shown in rodent models to be involved in obstruction-induced fibrosis, it is important to verify these in animal models with greater resemblance to the human kidney, such as the pig. As opposed to the unipapillary structure of the rodent kidney, the pig has multipapillary kidneys similar to the human. Furthermore, the porcine kidney has urine transport characteristics and predominantly short-looped nephrons like the human kidney ( 19, 41, 47 ), factors likely to be important when conditions of urinary flow hindrance are being studied.

The mechanisms leading to obstruction-induced fibrosis are complex and have been elucidated by both in vitro and in vivo studies ( 1, 20 ) but not previously in the neonatal porcine kidney. The processes of apoptosis, epithelial-mesenchymal transition and accumulation of activated fibroblasts contribute in parallel to the progression of renal fibrosis ( 1, 20 ). In vivo studies in rodent models have shown that ANG II, which is upregulated in the neonatal obstructed kidney, appears to play a key role in fibrosis formation ( 1, 31, 45 ). The mechanisms may be multifactorial. By inducing vasoconstriction via AT 1 receptors in presumably the efferent arterioles, consequential hypoxia of the tubulointerstitial compartment may stimulate fibroblast proliferation, extracellular matrix synthesis, and apoptosis ( 40 ). Furthermore, the AT 1 receptors play a role in the stimulation of a number of cytokines such as the profibrotic growth factor transforming growth factor- (TGF- ) ( 16, 56 ). The stimulatory impact of ANG II on this multitude of components in the cascade of events leading to fibrosis has given hope for the development of novel therapeutic approaches aiming at inhibiting the effect of ANG II. In a recent study of complete ureteral obstruction in adult rats, the ANG II antagonist losartan appeared to attenuate apoptosis and fibrosis, leading the authors to conclude that ANG II blockade may be valuable in the antifibrotic armentarium ( 33 ). Such a recommendation may, however, not necessarily be applicable to the situation of congenital partial obstruction, where ANG II is likely to play a different role and is an important constituent of normal renal development ( 27, 54 ).
9 k# ?4 T1 u$ F4 u! P
The purpose of the present study was to characterize apoptosis and fibrosis formation in a well-established neonatal pig model with PUUO induced during ongoing nephrogenesis. The role of ANG II was investigated by blocking the AT 1 receptor with candesartan, ( 28 ), after the end of nephrogenesis. To quantitate apoptosis, we used a specific technique with immunohistochemical staining for active caspase 3 specifically identifying the effector caspases only found in apoptotic cells ( 43 ). By use of stereological techniques, the absolute numbers of apoptotic cells in the interstitial compartment, in the glomeruli, and in the different tubular segments were counted ( 18 ). Distinguishing between different tubular segments and intrarenal capillaries often proves problematic. We therefore introduce a new variant of the physical fractionator involving a third immunostained section to localize the apoptotic cells to specific tubular segments. The extent of interstitial collagen accumulation was quantified using stereological techniques after visualization by picro Sirius red staining and further by hydroxyproline quantification.

MATERIALS AND METHODS% [* V, G* T1 t" S- [+ u

Experimental design. The experiments were performed in 30-day-old female Danish Landrace pigs who at age 2 days were subjected to partial obstruction of the left ureter using the psoas implantation method introduced by Ulm and Miller ( 51 ) or sham operation (previously described by us in detail) ( 23 ). In brief, under isoflurane anesthesia a 3- to 3.5-cm flank incision was made on the left side using sterile techniques. The muscle layers were divided, and the peritoneum was gently pushed medially. The left ureter was identified at the lower renal pole, and 3 cm were carefully mobilized, preserving the periureteral adventitial tissue. A 2- to 2.5-cm longitudinal slit was made in the adjacent psoas muscle. The obstruction was created embedding the mobilized ureter in the muscle slit and closing the muscle with three 5-0 nylon sutures. In the sham-operated groups, the ureter was exposed but not obstructed., L; M) r' A0 X5 J$ K, M

In one-half of the partial obstructed and in one-half of the sham-operated piglets, the AT 1 receptors were blocked using candesartan as follows. Before surgery, the piglets were randomized to one of four experimental groups of which siblings were equally distributed; sham operation (sham; n = 11), sham operation and candesartan treatment (sham can; n = 10), PUUO (PUUO; n = 12), and PUUO and candesartan treatment (PUUO can; n = 12). At age 23 days, a 2-ml osmotic minipump (Alzet Osmotic Pump, Durect, Cupertino, CA) was implanted subcutaneously under light ketamine sedation and subcutaneous lidocaine anesthesia in the groin of each piglet. In the sham can and PUUO can groups the pumps were filled with candesartan (CV-11974, kindly provided by AstraZeneca, Mölndal, Sweden) dissolved in 1 M Na 2 CO 3 and physiological saline releasing candesartan at 0.12 mg/h (corresponding to 0.03 mg·kg -1 ·h -1 ) for 7 days, balancing the maximal capacity of the osmotic pump with the expected arterial pressure response. Previous studies in adult rats demonstrated that a candesartan bolus injection of 0.01 mg/kg did not reduce arterial pressure but significantly changed renal functional parameters whereas doses of 0.1 mg/kg significantly decreased arterial pressure ( 6 ). During treatment with candesartan, arterial blood pressure was measured ambulatory and did not differ among groups ( 25 ). In the sham and PUUO groups, the osmotic pumps were filled with isotonic saline only. The study complied with the Danish regulations for the care and use of experimental animals and was approved by the Danish Animal Experiments Inspectorate.

Perfusion fixation. Kidneys were antegrade perfusion fixed under anesthesia with 4% phosphate-buffered formalin for a total of 5 min through the aorta from below the renal arteries ( 24 ) after a prerinse with phosphate buffer for 45 s. Before removal of the kidneys, the anesthetized pig was euthanized using potassium chloride. The kidneys were weighed with an empty pelvis and subsequently cut from top to bottom in 4-mm-thick horizontal sections. One-half of the sections were systematically and uniformly random sampled consistent with the fractionator principle ( 42 ) and stored overnight in 4% formaldehyde.' p0 n) h$ X9 ^, e" O. E

Stereology. Using systematic, uniform random sampling, a quarter of the kidney was sampled (block-sampling fraction), embedded in paraffin, and cut in 2.5-µm-thick sections using a calibrated microtome (Microm HM 355, Walldorf, Germany). Paraffin embedding can cause significant shrinkage of tissue. Therefore, the section-sampling fraction constituted the ratio between the shrunken and varying heights of the original cut 4-mm-thick sections and the microtome advance (MA) ( 21 ), which was equal to 2.5 µm. A third and final sampling step, the area-sampling fraction, was applied to the sampled sections as the ratio between the area of a 2-dimensional counting frame and the area delineated by the step lengths d x and d y using a motorized microscopic stage. In this final sampling fraction, the number of cells in question was quantitated using the physical disector probe principle ( 24, 42 )

where T * is the shrunken section thickness measured from several randomly sampled sections by serial sectioning and weighted by the number of cells, q -, counted in this tissue. The section number is denoted i and a (frame) is the area of the counting frame. Finally, Q - is the number of cells (nuclei) counted in the kidney. This number is divided by two, because the counting is performed both ways in the physical disector.* s+ j& f% B% `
4 W& a7 w2 B; J5 C
Tubular lengths were estimated in both cortex and medulla by counting the number of tubule profiles sampled by a 2-dimensional counting frame and estimating the length density, which is converted to absolute length by multiplying with the shrinkage-adjusted renal volume
5 E9 _3 u+ h2 C7 S! |
where Q (tube) is the number of tubule profiles, a (frame) is the area of the test frame, p is the number of test points, P (kidney) is the number of test points which hits kidney tissue, and the volume of the kidney is denoted V(kidney). The factor 2 is only valid when isotropic area probes are used, which is not the case in the medulla of the kidney. Isotropy means that all directions have equal probability of being chosen. This factor is therefore not correct in this study, and the total length results are biased with an unknown factor. This, however, applies to all four groups.6 S4 t- X0 m9 m# N3 l
- H( a7 G& v) ?3 c
Throughout this study, we cut the tissue samples in three adjacent sections of 2.5 µm of thickness. The first two sections were used for counting apoptotic cells (the physical disector probe), the last section for distinguishing between the thin limb of Henle and the capillaries, and the distal tubules and the collecting ducts.

With regard to tissue deformation (shrinkage), small kidney samples were weighed and the weight was converted to volume by multiplying using 1.04 cm 3 /g (volume before). The volume of kidney samples after paraffin embedding (volume after) was estimated by point counting and the principle of Cavalieri ( 42 ). Tissue deformation was equal to 1 - (volume after/volume before), which was accounted for.
- P5 [+ N( B: B$ y: e; `8 @) c. h& l
For all tissue preparation and stereological quantitation the observer was naive to the origin of the histological sections.

Immunohistochemistry and collagen assessment. Apoptotic cells were identified with the active caspase 3 technique using a rabbit polyclonal antibody specific for the activated subunit of caspase 3 (1:200, Cell Signalling Technologies, Beverly, MA) and affinity-purified biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA). Tubular segments were identified and separated by staining with aquaporin-2 (AQP2) antibodies for collecting duct principal cells (N-20, 1:50, Chemicon, AH Diagnostics, Aarhus, Denmark). The capillaries were identified using antibodies against von Willebrand factor after demasking with proteinase K (both from DakoCytomation, Glostrup, Denmark). The negative control included replacement of the primary antibodies with nonimmune sera, which showed no staining.

The extent of interstitial collagen accumulation was visualized in sections by picro Sirius red, which stains connective tissue fibers selectively ( 46 ).

Hydroxyproline content was measured in tissue samples from a midsection of the kidneys in the four experimental groups. The samples were collected and defatted in chloroform:methanol (2:1) including protease inhibitors for 21 h at 4°C. Then, 200 µl 6 N HCl were added to 10 mg of dried and pulverized tissue and hydrolyzed at 118°C for 18 h. This was neutralized using 120 µl 10 M sodium hydroxide. Hydroxyproline was measured in the supernatant after centrifugation as described elsewhere ( 53 ). The collagen content was calculated from total hydroxyproline using 7.46 as the correction factor and expressed as a proportion of the tissue dry weight (µg collagen /10 mg dry weight tissue).' I% z/ G6 r4 H  O
) ^4 I  o+ n! \1 Y
Statistics. Values are presented as mean (SD, [95% confidence interval]) and compared using ANOVA and the post hoc Bonferroni procedure. Data for tubular apoptosis, cell number, and lengths were compared using an unpaired Student's t -test. Values were considered statistically significant at P

RESULTS

All pigs had macroscopically normal-appearing left kidneys at study entry and right kidneys at study's end. All partially obstructed kidneys were found to have significant hydronephrosis at age 30 days. Seven animals were lost due to anesthesia-related death ( n = 3) or failure to thrive ( n = 4). In four animals, perfusion fixation was not possible for practical reasons, leaving a total of nine sham, nine sham can, seven PUUO, and nine PUUO can to be included in the study. Urine culture at 30 days did not demonstrate urinary tract infection.

Kidney weight and volume. Body weight did not differ among the experimental groups, demonstrating that somatic growth was unaffected by neonatal induced PUUO and by candesartan treatment ( Table 1 ). PUUO, on the other hand, significantly increased partially obstructed kidney wet weight but not kidney volume compared with sham-operated kidneys. Candesartan did not change kidney wet weight or volume in either partially obstructed or sham-operated kidneys compared with the saline-treated groups ( Table 1 ).3 @1 R$ i  @2 `* t) y7 ~) f/ q# Y

Table 1. Kidney wet weight and kidney volume in the 4 experimental groups
( u) j/ v0 F- E% n
PUUO increased interstitial cell apoptosis and fibrosis formation. The apoptotic interstitial cells were identified by histochemical staining for active caspase 3 as shown in Fig. 1 and quantitated using stereological techniques. Twenty-eight days of PUUO induced at age 2 days significantly increased the total number of apoptotic interstitial cells in the partially obstructed kidneys compared with sham-operated kidneys (4,568·10 4 (SD 3,606·10 4 [7,903·10 4, 1,234·10 4 ]) vs. 1,363·10 4 (SD 1,230·10 4 [2,309·10 4, 418·10 4 ]); P = 0.004) ( Figs. 1 and 2 ).
3 T5 F; n, P+ j# P3 N3 [9 G
Fig. 1. Immunohistochemical staining for active caspase 3 demonstrated an increase in the number of apoptotic interstitial cells (red staining cells, indicated by arrows) in partially obstructed kidneys [ C and E (high magnification of inset in C )]. In contrast, the number of apoptotic interstitial cells in the candesartan-treated partial obstructed kidneys [ D and F (high magnification of inset in D )] did not differ from sham-operated kidneys ( A and B ), where no apoptotic cells are visualized in these representative sections.7 L2 G8 [$ E, f6 @0 H( _  ]
. _6 Q  h! c) J3 g& H! Y
Fig. 2. The total number of apoptotic interstitial cells quantitated by active caspase 3 technique and stereology was significantly increased in the partially obstructed kidneys [unilateral partial ureteral obstruction (PUUO)] compared with sham-operated kidneys (* P = 0.004) and to candesartan-treated partially obstructed kidneys (PUUO can) (* P = 0.004)./ N( B* B! m& ^# |' y
- I) P, c- J& w1 d+ T, F
By use of stereological techniques and picro Sirius red staining for collagen fibers, the volume of fibrosis in the entire kidney could be estimated. The volume of fibrosis was significantly increased in the partially obstructed kidneys compared with sham-operated kidneys (4.62 (SD 2.43 [6.65, 2.59]) vs. 2.49 (SD 1.34 [3.51, 1.46]) cm 3, P = 0.03) ( Figs. 3 and 4 ). The increased fibrosis formation in the partially obstructed kidneys was confirmed by quantitation of hydroxyproline and calculation of the collagen content (per 10 mg of dry tissue) in a section from the middle part of the kidney. The collagen density in the partially obstructed kidneys was significantly increased compared with sham-operated kidneys (443.7 (SD 176.7 [663.1, 224.4]) vs. 208.0 (SD 28.0 [276.6, 139.4]) µg/10 mg dry tissue, P = 0.009), confirming the results from the whole-kidney stereological analysis ( Fig. 5 ).

Fig. 3. The total volume of fibrosis visualized by picro Sirius red staining for collagen fibers was significantly increased in PUUO kidneys ( C ) compared with sham-operated kidneys ( A ). Candesartan treatment did not reduce the PUUO-induced fibrosis ( D ) and did not induce fibrosis in sham-operated kidneys ( B ).

Fig. 4. The total volume of fibrosis quantitated by stereological methods in kidney sections stained with picro Sirius red for collagen fibers was significantly increased in PUUO kidneys compared with sham-operated kidneys (* P = 0.03).
2 P7 q3 d4 V$ D* Z4 k5 T  a5 E
Fig. 5. The collagen density calculated from hydroxyproline content in a midsection of the kidneys was significantly increased in PUUO kidneys compared with sham-operated kidneys (* P = 0.009).' O/ ]8 X8 p: C4 E: u
" J9 F" m" F- N3 T6 F0 ?7 |7 r
Tubular apoptosis was not increased after 28 days of PUUO. To quantify the number of apoptotic cells in the different tubular segments, a new technique was developed using an additional immunostained section for segmental identification ( Fig. 6 ). The results revealed that the number of apoptotic cells was not increased in the proximal tubules ( P = 0.76), in the distal tubules ( P = 0.29), or in the collecting ducts ( P = 0.15) compared with sham-operated kidneys after 28 days of partial obstruction ( Table 2 )." n7 Y+ P+ c  M# `6 l/ v% M
1 I6 ^' n- X) s' _- V! x$ u
Fig. 6. Immunohistochemical staining showing an apoptotic cell (red staining cell, arrow) in a proximal tubule ( A ), aquaporin-2 (AQP2; red apical staining, arrows) in collecting duct cells ( B ), and von Willebrand factor in capillaries (red staining, arrows, C and D ).9 E( z: N1 t  ]% @. ?- D4 ^. E
! S1 G. f4 Y0 q0 [. O' H
Table 2. Tubular cell apoptosis after 28 days of partial obstruction or sham operation
! H2 ^# Q* M# d% D* O; M+ J8 P
Since the apoptotic response of upper tract obstruction is dynamic ( 15 ), tubular apoptosis may have peaked at an earlier time point, subsequently leading to decreased tubular length and cell number. We therefore used stereological techniques to quantify the total tubular length and the total number of tubular cells in the left kidneys of saline-treated pigs. The results revealed that partial obstruction for 28 days did not lead to decreased length of the proximal tubules ( P = 0.13), the thin limb of Henle ( P = 0.23), the distal tubules ( P = 0.52), or the collecting ducts ( P = 0.23) compared with sham-operated rats ( Table 3 ). Furthermore, it did not lead to a decreased number of cells in the proximal tubules ( P = 0.49), in the thin limb of Henle ( P = 0.41), in the distal tubules ( P = 0.20), or in the collecting ducts ( P = 0.41) compared with sham-operated kidneys ( Table 4 )./ n% |. j+ _% L. h

Table 3. Tubular length after 28 days of partial obstruction or sham operation2 j3 [8 f+ ~! o

Table 4. Tubular cell number after 28 days of partial obstruction or sham operation
/ x4 L+ U) G3 L6 P: B
Candesartan prevented the PUUO-induced interstitial cell apoptosis but not fibrosis. To elucidate the role of AT 1 receptor stimulation in PUUO-induced interstitial fibrosis and apoptosis, one-half of the pigs were treated with candesartan from age 23 to 30 days. Candesartan treatment attenuated the PUUO-induced increase in interstitial apoptosis as the total number of apoptotic interstitial cells in these kidneys were not increased compared with sham-operated kidneys (1,749·10 4 (SD 818·10 4 [2,378·10 4, 1,121·10 4 ]) vs. 1,169·10 4 (SD 387·10 4 [1,466·10 4, 871·10 4 ]); P = 0.50). In accordance the number of apoptotic interstitial cells in the partial obstructed kidneys of candesartan-treated piglets was significantly decreased compared with partial obstructed kidneys from saline treated piglets (1,749·10 4 (SD 818·10 4 [2,378·10 4, 1,121·10 4 ]) vs. 4,568·10 4 (SD 3,606·10 4 [7,903·10 4, 1,234·10 4 ]; P = 0.004) ( Figs. 1 and 2 ).  r9 y9 d  g9 ^- R; M
9 v, M% \7 e3 |% r  {5 G8 D
Candesartan treatment did not reduce the PUUO-induced fibrosis formation as the total volume of fibrosis (estimated by stereological techniques) in the partial obstructed kidneys did not differ between candesartan-treated and saline-treated pigs (4.96 (SD 2.64 [7.17, 2.76]) vs. 4.62 (SD 2.43 [6.65, 2.59]) cm 3, P = 0.73) ( Figs. 3 and 4 ). The increased fibrosis formation in both candesartan-treated and saline-treated obstructed groups was confirmed by the collagen density, which did not differ between the two groups (375.9 (SD 116.7 [473.5, 278.3]) vs. 443.7 (SD 176.7 [663.1, 224.4]) µg/10 mg dry tissue, P = 0.62) ( Fig. 5 ). Candesartan did not induce fibrosis in the sham-operated kidneys (2.72 (SD 1.19 [3.63, 1.81]) cm 3 and did not increase the collagen density (200.5 (SD 130 [320.7, 80.3]) µg/10 mg dry tissue compared with saline treatment ( P = 0.70 and P = 0.89, respectively).# E% ]4 E4 o" R  A
8 z! Y% i5 Q7 J1 X: o, V& s' N* E
Candesartan prevented PUUO-induced glomerular apoptosis. Immunohistochemistry using caspase 3 antibodies ( Fig. 7 ) and stereological methods revealed that the total number of apoptotic glomerular cells was significantly increased in kidneys subjected to partial obstruction compared with sham-operated kidneys (6,775·10 3 (SD 3,786·10 3 [10,277·10 3, 3,274·10 3 ]) vs. 2,520·10 3 (SD 2,079·10 3 [4,119·10 3, 922·10 3 ]), P = 0.03) ( Fig. 8 ).

Fig. 7. Immunohistochemical staining for active caspase 3 demonstrating apoptotic glomerular cells (red staining cells in glomeruli, arrows) in a representative section from PUUO kidneys of saline-treated pigs ( B ) as opposed to glomeruli without apoptosis in representative sections from sham-operated kidneys of saline-treated pigs ( A ) and PUUO kidneys of candesartan-treated pigs ( C ).

Fig. 8. The number of glomerular apoptotic cells was significantly increased in PUUO kidneys compared with sham-operated kidneys (* P / ~0 G& C" A5 Q, ^+ z% E, Z, z' v) v
/ c* k& x* H7 z' g
Candesartan prevented PUUO-induced glomerular cell apoptosis, since the number of apoptotic glomerular cells did not differ between partially obstructed or sham-operated kidneys in candesartan-treated pigs (3,679·10 3 (SD 3,100·10 3 [6,062·10 3, 1,296·10 3 ]) vs. 2,635·10 3 (SD 2,194·10 3 [4,322·10 3, 949·10 3 ]), P = 0.42) ( Fig. 8 ).

DISCUSSION

In the present study, we used a well-established neonatal pig model with high analogy to the human kidney. For the first time, fibrosis formation and tubulointerstitial and glomerular cell apoptosis are quantitated in the multipapillary kidney subjected to chronic neonatal PUUO. The results demonstrated that chronic blockade of the AT 1 receptor by candesartan after completion of nephrogenesis prevented PUUO-induced interstitial apoptosis but not fibrosis. Furthermore, AT 1 receptor blockade prevented the increased glomerular apoptosis, demonstrating that ANG II plays a role in obstruction-induced apoptosis in this model.$ H3 i$ f: ~2 t
& s( m, l; B6 p* |
Identification of apoptotic renal cells. To quantify tubulointerstitial cell apoptosis, we used antibodies against activated caspase 3. The apoptotic event is initiated by one of several pathways converging into a common arm characterized by the orderly activation of a family of caspases acting as initiators, executioners, or cytokine processors, leading to the structural changes of apoptosis ( 43 ). Activated caspase 3 plays a central role in the execution and has proven a valid predictor of apoptosis ( 29, 55 ). The dynamic expression of caspases and the central role of caspase 3 have also been documented during urinary tract obstruction in mice and in human tubular cells exposed to mechanical stretch and stress ( 44, 49 ). We therefore chose to use identification of activated caspase 3 in the present study as this method specifically identifies cells undergoing apoptosis. This is opposed to the frequently used transferase-mediated dUTP-biotin nick end-labeling technique relying on DNA fragmentation, which is not specific for apoptotic cells. However, neither of these methods allows for characterization of the specific cell type undergoing apoptosis in the interstitial compartment.

The use of several antibodies and three parallel sections allowed for localization of the apoptotic cells to specific parts of the nephrons. For this purpose, we developed an improved staining technique using AQP2 antibodies to identify the collecting ducts and antibodies against von Willebrand factor to identify the capillaries. This ensured that each cell type was correctly identified and quantified./ ^! j/ M* t; J6 K8 t9 O$ {

To quantify the number of cells, a design-based stereological method (physical fractionator with varying sampling fraction) for the estimation of total numbers of "normal" and apoptotic cells was introduced. This physical fractionator with varying sampling fractions has been calibrated for tissue shrinkage, and the estimates are therefore independent of tissue deformation. To overcome the problem with estimating many different cell types in the same sampling procedure, counting frames with different sizes and varying step lengths were used in the CAST system. The aim was to make the error variance of the method (CE) so that the total variance (CV) would be less affected. In general, the aim is to design the study so that: 0.2
+ L: \$ ?$ m$ {" `7 _. R
Neonatal PUUO did not induce tubular apoptosis after 28 days of obstruction. We found that apoptosis in proximal or distal tubule cells or in collecting duct cells was not increased after 28 days of neonatal induced partial obstruction. Our results are supported by findings in the neonatal rat kidney where partial obstruction of 2-wk duration did not induce tubular cell apoptosis ( 48 ). This is opposed to models of complete ureteric occlusion, which have demonstrated increased tubular apoptosis that in the adult mouse model peaked after 2 wk of unilateral ureteral occlusion ( 15, 48 ). The rodent studies suggest that tubular apoptosis may appear in a dynamic expression related to the severity of obstruction. It is therefore possible that tubular apoptosis had occurred at an earlier time-point, i.e., before the investigation after 28 days of PUUO, or that the obstruction was not severe enough to induce tubular apoptosis in the present study. This is consistent with the absence of apoptosis in biopsy specimens from human hydronephrotic kidneys taken at the age of 9-16 mo ( 37 ).

If tubular apoptosis had peaked at an earlier time point in the present study, it could have led to increased cell destruction and thereby decreased tubular length. Because the kidneys in our study were perfusion fixed, which preserves structural integrity, tubular lengths could be quantified accurately. We found that neonatal induced PUUO did not compromise renal growth as indicated by kidney weight and volume and did not lead to decreased tubular lengths after 28 days of partial obstruction. These observations may reflect either insignificant tubular apoptosis or compensatory tubular proliferation as described in obstructed rat kidneys subjected to treatment with epidermal growth factor ( 11 ).

Interstitial cell apoptosis and fibrosis are increased by neonatal PUUO. Twenty-eight days of PUUO increased the total number of apoptotic interstitial cells in partially obstructed kidneys in this neonatal pig model. This finding is consistent with the increased apoptotic cell density and visual impression of increased apoptotic interstitial cell numbers in completely obstructed neonatal and adult rat kidneys ( 10, 12, 14 ). Using similar apoptosis-specific methods, the levels of apoptosis signaling molecules such as Fas and Fas ligand were elevated and associated with increased caspase 3 levels and interstitial apoptotic cells in adult mice with ureteral ligation, supporting the findings of the present study ( 15, 49 ).' c- G5 g1 ~/ k

Using picro Sirius red staining of collagen fibers and stereological techniques for whole-kidney quantification, we found that neonatal induced PUUO significantly increased interstitial fibrosis. The increased collagen density in the partially obstructed kidneys confirmed the increased amount of fibrotic tissue. Our findings are in accordance with the increased fibrotic tissue density found in neonatal rat kidneys with severe PUUO or temporary occlusion ( 13, 48 ) and in occluded adult rat kidneys ( 31 ). Moreover, the increased collagen density is also in agreement with observations in the occluded adult mouse kidney ( 17 ).
7 m4 a# T0 S* n% ~
In the present study and in previous rat studies of fibrosis and apoptosis, the harvested renal tissues are paraffin embedded before the staining procedures. The paraffin-embedding procedure is likely to induce varying degrees of tissue shrinkage and thereby different and unknown degrees of bias. We therefore used stereological methods calibrated for tissue shrinkage, providing estimates independently of tissue deformation. To our knowledge, such a correction has not previously been implemented in studies involving estimation of apoptotic cell or fibrosis densities in paraffin-embedded tissue from obstructed kidneys.

Candesartan prevented the PUUO-induced interstitial cell apoptosis but not fibrosis. Based on knowledge obtained from gene knockout animal models, tubular cell apoptosis is likely to play an important role in the complex process leading to interstitial fibrosis ( 1 ). However, the role of interstitial cell apoptosis in the fibrogenic response remains unclear. An important finding in the present study was therefore that the PUUO-induced interstitial cell apoptosis was prevented by candesartan treatment instituted after completion of nephrogenesis (which requires an intact ANG II response) ( 28 ) but the amount of fibrotic tissue was not reduced. The methods used did not allow for identification of the interstitial cell type undergoing apoptosis. A characteristic of the renal response to obstruction is the accumulation of inflammatory cells in the interstitium, releasing fibrogenic mediators ( 8 ). It may therefore be speculated that apoptosis clears inflammatory or other fibrogenic cells from the interstitium, thereby playing a salutary role in the fibrogenic response. However, despite the effect on apoptosis interstitial fibrosis was not changed by candesartan in the neonatal obstructed pig kidney. In previous studies using neonatal and grown rat models of complete unilateral obstruction, it was demonstrated that ANG II inhibition reduced fibrosis ( 30, 31, 35, 38, 39 ) but did not attenuate the increased interstitial apoptosis ( 14 ). Also, candesartan delivered by subcutaneous osmotic pumps in adult rat kidneys with complete obstruction for 28 days prevented interstitial fibrosis ( 52 ). Interestingly, in the present study we found that AT 1 receptor blockade reduced interstitial apoptosis but not fibrosis. It may be speculated that, in contrast to findings in the rat, the beneficial effect of ANG II inhibition on fibrosis formation was counterbalanced by the decreased removal of fibrogenic interstitial cells by apoptosis in the present model.
( a0 ~. E# t' X! G
The discrepancy with findings in the rat may be related to different obstruction models as complete occlusion of the ureter creates a situation that may not be comparable to the partial obstruction induced in the present study. It may also be related to the initiation of ANG II inhibition sooner after establishment of the obstruction in the rat models than in the present pig study. This is supported by a recent study of partial obstruction in neonatal rats, where angiotensin-converting enzyme inhibition by enalapril further aggravated interstitial collagen accumulation when treatment was instituted 10 days after obstruction was induced compared with kidneys subjected to early ANG II inhibition ( 7 ). Based on these considerations, the clinical potential of blocking the AT 1 receptors as suggested by some may therefore not be as beneficial for the obstructed human kidney in preventing fibrosis as previously thought.- k$ j, e5 Q. s! O8 O, N& ?: m

Our findings suggest that other fibrogenic pathways acting independently of the AT 1 receptors are of key importance in obstruction-induced fibrosis. This is also supported by findings in the neonatal mouse kidney, where complete absence of ANG II established by zero-copy of the angiotensinogen gene did not prevent interstitial fibrosis formation secondary to ureteral occlusion ( 26 ). TGF- is an important regulator of many of the processes leading to fibrosis formation, including apoptosis and epithelial-mesenchymal transition ( 1, 32, 36 ). The TGF- levels are increased in the obstructed kidney stimulated by the AT 1 receptor ( 1 ), and previous studies in adult obstructed rat kidneys indicate that AT 1 receptor blockade reduces fibrosis by downregulation of TGF- ( 30 ). However, ongoing studies in the present pig model suggest that candesartan may downregulate TGF- without affecting fibrosis, suggesting other fibrogenic pathways in the neonatal pig model.  c8 g  ]) @0 [% |. A$ A: l
: t, N' O, I8 ~' k- r% s
Neonatal PUUO increased glomerular apoptosis. We found a significantly increased number of glomerular apoptotic cells after 28 days of partial obstruction established during ongoing nephrogenesis in this multipapillary pig model. This suggests that increased apoptosis may play a role in the PUUO-induced loss of glomeruli as previously documented after 24 wk of partial obstruction in this model ( 24 ). The causative relationship is supported by the decrease in glomerular number occurring after end of nephrogenesis in the neonatal obstructed rat kidney, demonstrating loss of previously formed glomeruli ( 48 ).

Although only few studies exist for comparison, our findings are contradictory to rodent models where glomerular cells appear to be resistant to obstruction-induced apoptotic cell death. In adult rodent models with complete obstruction, glomerular apoptotic densities detected by the transferase-mediated dUTP-biotin nick end labeling technique were not increased ( 49, 50 ). In contrast, glomerular apoptotic densities were increased in neonatal mice with complete obstruction although the numbers did not reach statistical significance ( 5 ). The discrepancy may be explained by differences in animal species and the methods used. The renal maturational stage is also of importance as tubular apoptosis induced by ureteral occlusion is found to be twofold greater in the neonatal than in the adult rat kidney ( 9 ).

The finding of increased glomerular apoptosis was not reflected in changes in obstructed kidney glomerular filtration rate in these kidneys ( 25 ). We previously showed that a significant reduction in the number of glomeruli was coexistent with intact glomerular filtration rate and differential function values in this model ( 24 ). This supports the view that measurements of renal function may not reflect pathological mechanisms involved in congenital hydronephrosis. and a better understanding of these findings may lead to improved monitoring and treatment of human congenital hydronephrosis.
; C+ _' ^. ^- d/ ?+ v5 ]5 I
Chronic AT 1 receptor blockade prevented PUUO-induced glomerular apoptosis. Blockade of the AT 1 receptor by candesartan after the end of nephrogenesis prevented PUUO-induced apoptosis of glomerular cells, demonstrating proapoptotic actions of the AT 1 receptor in this model. It was not possible to identify the glomerular cell type undergoing apoptosis. Our findings are in line with observations in weanling rat PUUO kidneys, where glomerular damage in the form of proteinuria was suppressed by ANG II inhibition ( 2 ). The role of ANG II in obstruction-induced apoptosis is controversial, and the pathophysiological mechanisms are not yet fully elucidated. In neonatal rat kidneys subjected to 3 days of complete occlusion, ANG II increased tubular cell apoptosis through the AT 2 but not the AT 1 receptor ( 14 ). However, in vitro studies suggest that both the AT 1 and the AT 2 receptors are involved in ANG II-induced glomerular epithelial cell and tubular cell apoptosis whereas only the AT 1 receptor mediates apoptosis of glomerular podocytes subjected to mechanical strain ( 4, 18, 22 ). These results demonstrate that ANG II mediates renal cell apoptosis by complex mechanisms and that species- and perhaps cell-specific differences exist. Experimental models with high analogy to the human kidney are therefore necessary to fully elucidate the pathophysiology of congenital hydronephrosis in children and thereby identify potential targets for preventing glomerular apoptosis and destruction.( ?! o2 ^1 `# m' x8 x
8 V, |5 r+ D, ?
In conclusion, neonatal induced PUUO leads to increased interstitial and glomerular cell apoptosis and fibrosis in this pig model with high resemblance to the human kidney. Candesartan treatment prevented the increased interstitial and glomerular cell apoptosis but not fibrosis formation. This suggests that pathways not involving AT 1 receptor stimulation contribute to neonatal obstruction-induced fibrosis or that prevention of interstitial cell apoptosis counteracts a potential antifibrotic effect of AT 1 receptor blockade in this pig model of congenital obstructive nephropathy. These observations may have implications for decision making and therapeutic considerations in children.$ n1 v9 h% n9 k9 A  m
2 H- c8 ~0 [# h+ x3 F
GRANTS7 K/ W. P7 _, }$ o6 i! j. y

The study was supported by the A. P. Moeller and Chastine Mc-Kinney Moellers Foundation, the Helen and Ejnar Bjørnow's Foundation, The Danish Medical Society, The Dagmar Marshalls Foundation, The Society of Congenital Bladder Extrophy, the Ib Henriksens Foundation, The Danish Medical Research Council, The University of Aarhus, The Novo Nordic Foundation, The Eva and Henry Fraenkels Foundation, The Alice Brenaa Foundation, and The Foundation of 17.12.1981. The MIND Centre at the University of Aarhus is supported by the Lundbeck Foundation. The Water and Salt Research Centre at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond).$ U1 `& ~5 s/ p, k

ACKNOWLEDGMENTS

The authors thank Gitte Kall, Majbritt Lundorff, Lone Lysgaard, Marianne Jensen, Lotte Arentoft, Ken Kragsfeldt, and Albert O. Meier for expert technical assistance. Associate Professor Mie Br el is thanked for providing the picro Sirius red staining.- u: C# k/ T: x3 d8 L
      【参考文献】
Bascands JL, Schanstra JP. Obstructive nephropathy: insights from genetically engineered animals. Kidney Int 68: 925-937, 2005.

  W& Q, h1 B) J6 c" c  M
* J+ G! [# [2 G5 A8 I& `
Beharrie A, Franc-Guimond J, Rodriguez MM, Au J, Zilleruelo G, Abitbol CL. A functional immature model of chronic partial ureteral obstruction. Kidney Int 65: 1155-1161, 2004.

5 F: r7 h* L( K4 D$ E0 e

Benfield MR, McDonald RA, Bartosh S, Ho PL, Harmon W. Changing trends in pediatric transplantation: 2001 Annual Report of the North American Pediatric Renal Transplant Cooperative Study. Pediatr Transplant 7: 321-335, 2003.7 T( x7 R. q! E7 n9 v( e

, P0 }0 \% ?7 D3 s; V$ N

Bhaskaran M, Reddy K, Radhakrishanan N, Franki N, Ding G, Singhal PC. Angiotensin II induces apoptosis in renal proximal tubular cells. Am J Physiol Renal Physiol 284: F955-F965, 2003.
2 P& l! D/ r- |

Cachat F, Lange-Sperandio B, Chang AY, Kiley SC, Thornhill BA, Forbes MS, Chevalier RL. Ureteral obstruction in neonatal mice elicits segment-specific tubular cell responses leading to nephron loss. Kidney Int 63: 564-575, 2003.: @7 t6 y: w3 Y
5 k6 C# s; W+ L! H9 l

Cervenka L, Wang CT, Navar LG. Effects of acute AT1 receptor blockade by candesartan on arterial pressure and renal function in rats. Am J Physiol Renal Physiol 274: F940-F945, 1998.1 f0 ^, a9 @. O8 Y2 u2 B% B

2 Z- F3 K' Y# L% y. x/ M
' b) C6 G7 q; |/ r, m
Chen CO, Park MH, Forbes MS, Thornhill BA, Kiley S, Yoo KH, Chevalier RL. Angiotensin-converting enzyme inhibition aggravates renal interstitial injury resulting from partial unilateral ureteral obstruction in the neonatal rat. Am J Physiol Renal Physiol 292: F946-F955, 2007.) i. e  p) L# f6 o8 Y, A" A" W9 }3 ~
! \' f: _4 H5 d3 f# c( y

Chevalier RL. Pathogenesis of renal injury in obstructive uropathy. Curr Opin Pediatr 18: 153-160, 2006./ [. z$ |' n( P6 P" {
  F9 |; @; n1 \. R7 j$ b- \2 Q

2 ?9 C  }& i1 T, ~2 D' X7 d* q
Chevalier RL, Chung KH, Smith CD, Ficenec M, Gomez RA. Renal apoptosis and clusterin following ureteral obstruction: the role of maturation. J Urol 156: 1474-1479, 1996.

Chevalier RL, Goyal S, Kim A, Chang AY, Landau D, LeRoith D. Renal tubulointerstitial injury from ureteral obstruction in the neonatal rat is attenuated by IGF-1. Kidney Int 57: 882-890, 2000.( ]0 N' N% ^, N2 G' y" P
; P+ @& A  \, {0 L* U  K

% [& K0 J* ]& d$ O. G; C; l4 P
Chevalier RL, Goyal S, Wolstenholme JT, Thornhill BA. Obstructive nephropathy in the neonatal rat is attenuated by epidermal growth factor. Kidney Int 54: 38-47, 1998.

Chevalier RL, Smith CD, Wolstenholme J, Krajewski S, Reed JC. Chronic ureteral obstruction in the rat suppresses renal tubular Bcl-2 and stimulates apoptosis. Exp Nephrol 8: 115-122, 2000.

' G4 ]  P$ t, f. u- k$ G
Chevalier RL, Thornhill BA, Chang AY. Unilateral ureteral obstruction in neonatal rats leads to renal insufficiency in adulthood. Kidney Int 58: 1987-1995, 2000.

6 z, e" q2 x1 x. W+ h
Chevalier RL, Thornhill BA, Wolstenholme JT. Renal cellular response to ureteral obstruction: role of maturation and angiotensin II. Am J Physiol Renal Physiol 277: F41-F47, 1999.( F7 x# W# P+ H( C% S# U* ?# P4 y

! n; i+ a5 A  P& Z. y

Choi YJ, Baranowska-Daca E, Nguyen V, Koji T, Ballantyne CM, Sheikh-Hamad D, Suki WN, Truong LD. Mechanism of chronic obstructive uropathy: increased expression of apoptosis-promoting molecules. Kidney Int 58: 1481-1491, 2000.4 h* u; _7 O4 h* [

: E4 E' x+ a9 P" ?$ R0 o

Chung KH, Gomez RA, Chevalier RL. Regulation of renal growth factors and clusterin by AT1 receptors during neonatal ureteral obstruction. Am J Physiol Renal Fluid Electrolyte Physiol 268: F1117-F1123, 1995.! ]1 Q& y! N  t  H! T# ^6 S

; R2 \6 a* l; H0 X
Cochrane AL, Kett MM, Samuel CS, Campanale NV, Anderson WP, Hume DA, Little MH, Bertram JF, Ricardo SD. Renal structural and functional repair in a mouse model of reversal of ureteral obstruction. J Am Soc Nephrol 16: 3623-3630, 2005.' F& ?; Z, j; X& K- Z/ B
' k0 Q, r; G' r3 C" ~& Q2 ?
* H: x, H- p. h

Ding G, Reddy K, Kapasi AA, Franki N, Gibbons N, Kasinath BS, Singhal PC. Angiotensin II induces apoptosis in rat glomerular epithelial cells. Am J Physiol Renal Physiol 283: F173-F180, 2002.. K9 F  q1 t6 H$ L# W+ Z

" z' w0 ~& R8 V  q0 T
Djurhuus JC, Frøkiær J, Munch JT, Knudsen L, Pham T, Constantinou CE. Regulation of renal pelvic pressure by diuresis and micturition. Am J Physiol Regul Integr Comp Physiol 259: R637-R644, 1990.
" ?  m# v! v2 J9 |7 S

: D) ]. l+ v6 z4 L1 [
Docherty NG, O'Sullivan OE, Healy DA, Fitzpatrick JM, Watson RW. Evidence that inhibition of tubular cell apoptosis protects against renal damage and development of fibrosis following ureteric obstruction. Am J Physiol Renal Physiol 290: F4-F13, 2006.

: i+ N% v1 _+ }( B- {; Y; d
Dorph-Petersen KA, Nyengaard JR, Gundersen HJ. Tissue shrinkage and unbiased stereological estimation of particle number and size. J Microsc 204: 232-246, 2001.
5 _0 H9 I& U8 g6 P

Durvasula RV, Petermann AT, Hiromura K, Blonski M, Pippin J, Mundel P, Pichler R, Griffin S, Couser WG, Shankland SJ. Activation of a local tissue angiotensin system in podocytes by mechanical strain. Kidney Int 65: 30-39, 2004.5 c$ ^) E$ H) R- s. D/ x
) m* l. {1 f$ t6 M. P

Eskild-Jensen A, Christensen H, Lindvig M, Frokiaer J, Rehling M, Jorgensen HS, Djurhuus JC, Jorgensen TM. Renal functional outcome in unilateral hydronephrosis in newborn pigs. J Urol 163: 1896-1900, 2000.

1 R# F( C3 G! q
Eskild-Jensen A, Frokiaer J, Djurhuus JC, Jorgensen TM, Nyengaard JR. Reduced number of glomeruli in kidneys with neonatally induced partial ureteropelvic obstruction in pigs. J Urol 167: 1435-1439, 2002.
) i" h3 B8 v/ V% D6 t6 x' R2 F

1 h# O8 I7 O4 g* L5 S- `* ~
Eskild-Jensen A, Thomsen K, Rungoe C, Ferreira LS, Paulsen L, Rawashdeh YF, Nyengaard JR, Nielsen S, Djurhuus JC, Frøkiær J. Glomerular and tubular function during AT1 receptor blockade in pigs with neonatal induced partial ureteropelvic obstruction. Am J Physiol Renal Physiol 292: F921-F929, 2007.) b" `# I3 A1 f, N1 T( J  F
, h1 i5 }, Z3 N3 g7 g5 L3 ^
8 X6 ?5 [6 s9 [0 E7 d, |% |
  Z1 p5 T/ H5 o2 o+ ^: O
Fern RJ, Yesko CM, Thornhill BA, Kim HS, Smithies O, Chevalier RL. Reduced angiotensinogen expression attenuates renal interstitial fibrosis in obstructive nephropathy in mice. J Clin Invest 103: 39-46, 1999.! v5 u9 p2 @2 j0 `2 Q4 X% s; ]1 Q0 g% J, U% U
& u" g+ t" _) \2 H

Gomez RA. Role of angiotensin in renal vascular development. Kidney Int Suppl 67: S12-S16, 1998.
) n4 ?- j% g$ z9 I9 U: D8 Q. W
2 D. _7 b+ j& `9 B: a, D1 R- M
( @# C4 o& v5 q1 s' C
Guron G, Friberg P. An intact renin-angiotensin system is a prerequisite for normal renal development. J Hypertens 18: 123-137, 2000.# W; |  h1 C+ h9 t. ?

9 E( Q& Q- r% L8 _- W2 Y! X

Hayashi M, Araki T. Caspase in renal development. Nephrol Dial Transplant 17, Suppl 9: 8-10, 2002.
; q+ d  l4 @- @3 R  ~
. Z/ H0 s" F  o/ X, B; o

Ishidoya S, Morrissey J, McCracken R, Klahr S. Delayed treatment with enalapril halts tubulointerstitial fibrosis in rats with obstructive nephropathy. Kidney Int 49: 1110-1119, 1996.
( o' `& `6 k1 Y  X" U0 m

Ishidoya S, Morrissey J, McCracken R, Reyes A, Klahr S. Angiotensin II receptor antagonist ameliorates renal tubulointerstitial fibrosis caused by unilateral ureteral obstruction. Kidney Int 47: 1285-1294, 1995." g! c4 _% h4 C) }0 i1 d$ c, v9 g

, S3 E- q/ b& e7 W
( Q9 N, B6 u. }- U) ^
Iwano M, Neilson EG. Mechanisms of tubulointerstitial fibrosis. Curr Opin Nephrol Hypertens 13: 279-284, 2004., O$ m* U( N7 w+ }/ O

Kellner D, Chen J, Richardson I, Seshan SV, El CM, Vaughan ED Jr, Poppas D, Felsen D. Angiotensin receptor blockade decreases fibrosis and fibroblast expression in a rat model of unilateral ureteral obstruction. J Urol 176: 806-812, 2006.

Kiley SC, Thornhill BA, Belyea BC, Neale K, Forbes MS, Luetteke NC, Lee DC, Chevalier RL. Epidermal growth factor potentiates renal cell death in hydronephrotic neonatal mice, but cell survival in rats. Kidney Int 68: 504-514, 2005.
4 v4 G! ]  L0 A6 N+ {- t! c8 Z

. R7 L4 u4 d) _6 c$ n
Klahr S, Ishidoya S, Morrissey J. Role of angiotensin II in the tubulointerstitial fibrosis of obstructive nephropathy. Am J Kidney Dis 26: 141-146, 1995.

! X  \& y# C/ j7 z/ S4 B

Klahr S, Morrissey J. Obstructive nephropathy and renal fibrosis. Am J Physiol Renal Physiol 283: F861-F875, 2002.# h- l0 g; B& r# e' M6 p7 W& V! W

2 o" }& ]) b% j

Lama G, Ferraraccio F, Iaccarino F, Luongo I, Marte A, Rambaldi PF, Esposito-Salsano M. Pelviureteral junction obstruction: correlation of renal cell apoptosis and differential renal function. J Urol 169: 2335-2338, 2003.% Q& j4 A: j( a! _+ S1 D5 }. c
& c# K: `7 P* O8 E7 L# N" _

Manucha W, Oliveros L, Carrizo L, Seltzer A, Valles P. Losartan modulation on NOS isoforms and COX-2 expression in early renal fibrogenesis in unilateral obstruction. Kidney Int 65: 2091-2107, 2004.- l5 y& J# w4 N* r0 h2 Q

Morrissey JJ, Ishidoya S, McCracken R, Klahr S. The effect of ACE inhibitors on the expression of matrix genes and the role of p53 and p21 (WAF1) in experimental renal fibrosis. Kidney Int Suppl 54: S83-S87, 1996.

: r  D4 _& w9 u
Nangaku M. Chronic hypoxia and tubulointerstitial injury: a final common pathway to end-stage renal failure. J Am Soc Nephrol 17: 17-25, 2006.; X$ p( [" `; ]
! `) J0 ~, _" E7 T7 m/ m, q

Nielsen TW, Maaske CA, Booth NH. Some comparative aspects of porcine renal function. In: Swine in Biomedical Research, edited by Bukstad LK and McClellan RO. Seattle, WA: Frayn, 1965, p. 529-537.. P5 u1 V9 e$ x, a5 z

8 @& U& e* u* ~* x, X: [# c) e
& @/ W) l) j0 O  P* d
Nyengaard JR. Stereologic methods and their application in kidney research. J Am Soc Nephrol 10: 1100-1123, 1999.* J' Z3 l- W- e5 h' Y6 E* i7 h* G, k

& G: M2 z% u* [! x) _% H
7 F5 `# a, H' B8 |
Philchenkov A. Caspases: potential targets for regulating cell death. J Cell Mol Med 8: 432-444, 2004.

+ ]6 K. _3 b  [. f1 j  E8 [/ h& f" c9 a

Power RE, Doyle BT, Higgins D, Brady HR, Fitzpatrick JM, Watson RW. Mechanical deformation induced apoptosis in human proximal renal tubular epithelial cells is caspase dependent. J Urol 171: 457-461, 2004.; G3 n; S. d% M' p2 p6 A
6 S5 k; r& h. A0 ?3 O. I4 V
9 g  X* \* v0 d% {1 k0 p( H
* G& N: i  q: }& ~- @- [% G6 B
Satoh M, Kashihara N, Yamasaki Y, Maruyama K, Okamoto K, Maeshima Y, Sugiyama H, Sugaya T, Murakami K, Makino H. Renal interstitial fibrosis is reduced in angiotensin II type 1a receptor-deficient mice. J Am Soc Nephrol 12: 317-325, 2001.

9 F4 c9 T& d$ g4 ^5 c
Sweat F, Puchtler H, Rosenthal SI. Sirius red F3BA as a stain for connective tissue. Arch Pathol 78: 69-72, 1964.4 W  z1 S  ~9 g& R
* y$ t8 A: p- i1 L4 c7 x( H

Terris JM. Swine as a model in renal physiology and nephrology: an overview. In: Swine in Biomedical Research, edited by Tumbleson M. New York: Plenum, 1986, p. 1673-1709.4 c5 u- F  ]8 F

! C! U) G6 C5 E
Thornhill BA, Burt LE, Chen C, Forbes MS, Chevalier RL. Variable chronic partial ureteral obstruction in the neonatal rat: a new model of ureteropelvic junction obstruction. Kidney Int 67: 42-52, 2005.4 [) B. V/ S  O3 g" H) ]
' M+ S! w* {4 s( _5 v( Z' F
! P7 [0 \# ?' m2 p6 g- ?, c
) b4 S- I9 |, q: @% K
Truong LD, Choi YJ, Tsao CC, Ayala G, Sheikh-Hamad D, Nassar G, Suki WN. Renal cell apoptosis in chronic obstructive uropathy: the roles of caspases. Kidney Int 60: 924-934, 2001.

# Q8 b* p4 m0 q% x' i
. n  I# M. I# G1 |: @
Truong LD, Petrusevska G, Yang G, Gurpinar T, Shappell S, Lechago J, Rouse D, Suki WN. Cell apoptosis and proliferation in experimental chronic obstructive uropathy. Kidney Int 50: 200-207, 1996.
0 Z: s5 h/ A$ s0 d- b
' w" e6 Y: @- t
* J" t$ K9 T. [' p* c/ r4 d
Ulm AH, Miller F. An operation to produce experimental reversible hydronephrosis in dogs. J Urol 88: 337-341, 1962." _, t# J6 V' o) N6 E! l
5 T7 z  `! u. V0 p# b1 }
! K! u( j( o7 ]; C: r

Wamsley-Davis A, Padda R, Truong LD, Tsao CC, Zhang P, Sheikh-Hamad D. AT 1A -mediated activation of kidney JNK1 and SMAD2 in obstructive uropathy: preservation of kidney tissue mass using candesartan. Am J Physiol Renal Physiol 287: F474-F480, 2004.
1 S- B( R8 k; ^

) l! o/ O* m1 u
Woessner JF. Determination of hydroxyproline in connective tissue research. In: The Methodology of Connective Tissue Research, edited by Hall DA. Oxford, UK: Joynson-Bruvers, 1976, p. 227-233." C4 a% U6 P+ _7 t

( H; X. `' X+ R% q, z- K3 y* @9 \
Wolf G. Angiotensin II and tubular development. Nephrol Dial Transplant 17, Suppl 9: 48-51, 2002.

9 |4 B, E7 f  P; ]5 |7 h
8 J7 b& K; x4 l4 _+ m9 `% o
Yang B, El Nahas AM, Thomas GL, Haylor JL, Watson PF, Wagner B, Johnson TS. Caspase-3 and apoptosis in experimental chronic renal scarring. Kidney Int 60: 1765-1776, 2001.

Yoo KH, Thornhill BA, Chevalier RL. Angiotensin stimulates TGF- 1 and clusterin in the hydronephrotic neonatal rat kidney. Am J Physiol Regul Integr Comp Physiol 278: R640-R645, 2000.

收藏0 分享0 顶0 踩0