Novel cystogenic role of basic fibroblast growth factor in developing rodent kid - 《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 ... 25 下一页

返回列表

查看: 426084\|回复: 249	go Novel cystogenic role of basic fibroblast growth factor in developing rodent kid [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 08:39 |只看该作者 |倒序浏览 |打印

作者：Zhuangwu Li,, Marina Jerebtsova,,, Xue-Hui Liu, Pingtao Tang, and Patricio E. Ray,,作者单位：1 Division of Nephrology, and 2 Center for Genetic Medicine, Children‘s Research Institute, Children‘s National Medical Center, and 3 The George Washington University, Washington, District of Columbia
   ) s) o2 G$ x6 K* z
   5 B7 h+ C$ ~1 T6 [



         / ?7 j! [- A7 t" n


                  / B& r" H9 [9 ^9 T

      / d+ ]& s  @. A% }! B; V

      【摘要】
   Basic fibroblast growth factor (bFGF) is a heparin-binding growth factor that is accumulated in human dysplastic and cystic renal diseases. Previous studies have shown that bFGF can modulate the growth of developing renal tubules; however, its role in the pathogenesis of renal cyst formation is not clearly understood. Here, we tested the hypothesis that overexpression of bFGF in developing rodent kidneys induces cyst formation in vivo. We used two different adenoviral-mediated gene-transferring approaches to overexpress bFGF in developing rodent kidneys. Initially, metanephric kidney (MK) explants harvested from embryonic day 15 Sprague-Dawley rats were infected with adenoviral vectors (rAd) encoding human bFGF or LacZ genes and transplanted under the renal capsule of adult female rats. Subsequently, to determine whether bFGF could induce renal cysts in developing kidneys with an intact renal collecting system, we injected rAd-bFGF or LacZ vectors in the retroorbital plexus of newborn mice. Basic FGF induced a more efficient integration of the MK explants into the host kidneys and increased the vascularization and proliferation of developing tubules, leading to tubular dilatation and rapid formation of renal cysts. In addition, we successfully expressed human bFGF in the kidney of newborn mice in vivo and induced tubular dilatation and renal cysts. In contrast, mice injected with rAd- lacZ did not develop tubular dilatation or renal cysts. To the best of our knowledge, these experiments show for the first time that overexpression of bFGF in developing rodent kidneys can induce the formation of renal cysts in vivo. ( T$ c& x9 x; B" W  _
      【关键词】 renal development adenoviralmediated gene transferring human immunodeficiency virusnephropathy
   BASIC FIBROBLAST GROWTH FACTOR (bFGF) is a potent angiogenic growth factor belonging to the family of heparin-binding growth factors (FGFs) ( 1 ). Basic FGF lacks a conventional signal sequence for secretion and in normal conditions is stored as an inactive form in the vessel walls and basement membranes bound to heparan sulfate proteoglycans (HSPG) ( 22 ). In this manner, HSPG functions as a bioactive reservoir of bFGF and can regulate the release and mitogenic activity of bFGF during the processes of renal tubular growth and development ( 21, 31 ). In support of this notion, previous studies have shown that HSPG and heparin can modulate the mitogenic activity of bFGF in cultured renal fibroblasts and epithelial cells ( 3, 13 ). Moreover, bFGF is accumulated in human dysplastic kidneys with increased mesenchyma ( 30 ), and in adult kidneys with renal interstitial fibrosis ( 19, 32 ), where it can induce the transdifferentiation of renal tubular epithelial cells into fibroblasts ( 33 ). Taken together, these studies have established a clear role of bFGF in the pathogenesis of renal fibrosis in both rodent and human kidneys.

Less well understood, however, is the role that bFGF may have in the development of renal cysts. Gupta et al. ( 9 ) reported high levels of bFGF in the urine of a child with autosomal recessive polycystic kidney disease and suggested that bFGF may be a useful noninvasive marker to monitor the progression of cystic renal diseases in children. We have found that bFGF is accumulated bound to HSPG in the renal tubules and interstitium of human immunodeficiency virus (HIV)-transgenic mice/rats and HIV-infected children with renal diseases characterized by the formation of renal microcysts ( 16, 24 - 26 ). In these renal diseases, bFGF can be released from the renal extracellular matrix, leading to the proliferation of tubular epithelial cells and renal fibroblasts ( 3, 9, 16 ). In support of this notion, previous studies have shown that bFGF can induce the proliferation of renal tubular epithelial cells harvested from the urine of children with HIV-associated nephropathy (HIVAN) ( 16, 26 ). Thus there is enough clinical evidence to suggest that the accumulation of bFGF in HIV-infected children may facilitate the development of renal cysts by inducing abnormal proliferation of renal tubular epithelial cells. Here, we tested the hypothesis that overexpression of bFGF in vivo induces renal cysts in developing rodent kidneys. We used two novel adenoviral-mediated renal gene-transferring approaches recently developed in our laboratory to overexpress bFGF in vivo in developing rodent kidneys. To the best of our knowledge, these findings demonstrate for the first time that bFGF can induce the formation of renal cysts in developing rodent kidneys in vivo.6 {3 V) C# a: Z# u1 Z+ c

MATERIALS AND METHODS
; m- S6 _& L5 O5 _1 t
Metanephric kidney whole organ cultures. All animal experiments were carried out according to the institutional guidelines for animal use. The metanephric kidney (MK) explants were surgically dissected from embryonic day 15 Sprague-Dawley rats as described by Rogers et al. ( 28 ) and placed on sterile membranes from Nuclepore (110409, Pleasanton, CA) mounted on Falcon plastic dishes. Subsequently, they were cultured in DMEM-Ham's F-12 from Biofluids (Rockville, MD) with Ricther's modification from Life Technologies (Gaithersburg, MD). The culture media were also supplemented with 50 µg/ml transferrin, 1 mM HEPES, 2 mM L -glutamine, 5 µg/ml insulin, and 5 x 10 -8 M dexamethasone, all purchased from Sigma (St. Louis, MO).
7 r$ R6 q8 N) X! _
Treatment of MK explants with recombinant adenoviral vectors. The E1-deleted recombinant adenoviral (rAd) carrying the Escherichia coli lacZ gene encoding - galactosidase (designated rAd-lacZ) was generated as described previously by Kozarsky et al. ( 15 ). In this viral vector, lac Z expression is controlled by the cytomegalovirus (CMV) enhancer and the chicken -actin gene promoter. The particle/plaque-forming unit (pfu) ratio of the virus stock used in these experiments was 100. The E1-deleted rAd vector carrying the cDNA containing the 700-bp sequence encoding the low-molecular-weight (18 kDa) form of wild-type human bFGF under the control of the CMV enhancer/promoter sequence and the SV40 poly(A) sequence (designated rAd-bFGF) was generated by Gupta et al. ( 8 ). All MK explants were exposed to either rAd-lacZ or rAd-bFGF vectors (5 x 10 7 pfu/ml) for 4-12 h and followed in vitro for an additional 48-72 h. In addition, to validate our experimental rAd-system, additional MK explants ( n = 6 per group) were cultured in the presence of PBS (20 µl), or human recombinant bFGF (hrbFGF) from Biosource International (Caramillo, CA) at 20 ng/ml, alone or in combination with bFGF neutralizing antibodies (30 µg/ml bFGF antibody from R&D Systems, Minneapolis, MN; or 1:20 dilution of a rabbit polyclonal bFGF antibody, gift from Dr. A. Baird, PRIZM, Pharmaceuticals, San Diego, CA) for 48 h ( 7 ). The number of collecting duct branches and developing epithelial cells showing positive staining for the Dolichos biflorus agglutinin (DBA) was counted in five randomly selected microscopic fields ( x 25) in each section, using computer-assisted image-analysis software (Optimas v6.2; Media Cybernetics, Silver Spring, MD).

Immunohistochemical studies. Embryonic kidney explants were washed in PBS (pH 7.4), fixed in 10% neutral formalin, and embedded in paraffin. Renal sections were cut at 4 µm, deparaffinized, and rehydrated. The sections were routinely stained with hematoxylin for qualitative and quantitative analyses. Endogenous peroxidase activity was blocked by treating sections with 3% H 2 O 2 in 100% methanol for 10 min, and the immunostaining was performed with a commercial streptavidin-biotin-peroxidase complex Histostain SP kit from Zymed (South San Francisco, CA) as previously described ( 12 ). The following markers of cell differentiation and growth were used: DBA (Vector Laboratories, Burlingame, CA), to detect ureteric bud and developing collecting duct cells; a polyclonal rabbit antibody against the von Willebrand factor (vWF) from Dako (Capinteria, CA) to detect endothelial cells; the ED-1 antibody to detect rat mononuclear cells (Dako); the proliferating cell nuclear antigen antibody (PCNA) from Zymed; and the phospho-p44/42 MAPK (9106 and 9101) antibodies from Cell Signaling Technology (Beverly, MA) to detect a signaling target of bFGF. Basic FGF was identified using affinity-purified IgG fractions (2.5 µg/ml) from a rabbit polyclonal antibody directed against a unique peptide sequence of bFGF ( 7 ). Controls included replacing the primary antibody with equivalent concentrations of nonimmune IgG and omitting the first antibodies. The immunohistochemical results were quantitated by counting all cells stained positive in five microscopic fields at x 25-40 magnification, using the computerized program described above. Results were expressed as means ± SE and reported as percentage of cells stained positive for each specific marker relatively to the control sections.
) v6 r' L/ K- ]3 |8 @5 C! t
Transplantation of rat MK explants. Whole MK explants were dissected from E-15 Sprague-Dawley rat embryos and cultured on 3.0-µm collagen-coated sterile membranes as described above. Subsequently, 10 MK explants in each group were exposed to either rAd- lacZ or rAd-bFGF vectors (5 x 10 7 pfu per ml) for 4 h and implanted within 30 min under the renal capsule of anesthetized 6-wk-old female Sprague-Dawley rats ( n = 6) as described before ( 27 ). Seven to 15 days after transplantation, all rats were killed with CO 2, and their kidneys were harvested for further histological analysis. To document the expression of bFGF and lacZ on the MK explants, six additional E15 MK explants in each group were transduced with rAd- LacZ or rAd-bFGF vectors and cultured in vitro for 48-72 h.' {5 g! F7 I; S0 H/ m  K* P

Injection of rAd-bFGF into newborn mice. C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). One-day-old pups were injected via the retroorbital vein plexus with 2 x 10 9 particles/g body wt of rAd-bFGF, rAd- lacZ, or with 20 µl PBS (sham infections; n = 4 in each group). In preliminary experiments, we detected the peak levels of bFGF production at approximately 5 to 10 days after the rAd-bFGF injection. Thus all mice were killed 10 days after the adenoviral infection. To determine whether renal cells from newborn mice could be also successfully transduced via a retroorbital injection of rAd-bFGF, four adult mice were injected with a similar dose of rAd-bFGF as described above. Changes in renal tubular dilatation were graded in a blinded manner as follows: grade 0: no tubular changes; grade 1:
( S$ `  Y3 T. W/ ~2 n$ c$ U8 J
RT-PCR. Total kidney RNA was extracted using the TRIzol reagent from Life Technologies. RNA was reverse transcribed to cDNA using the SuperScript RT system with random oligomer primers (Life Technologies) and amplified by PCR for 30 cycles (94°C 30 s, 55°C 30 s, and 72°C 45 s) using specific human buff primers (5'-CATGGCAGCCGGGAGCATCACC and 3'-TCAGCTCTTAGCAGACATTGG). To prevent a false positive result due to the PCR amplification of the episomal adenoviral DNA, additional control samples were done in which the reverse transcription mix was omitted. RT-PCR studies with mouse GAPDH primers were done as a control for sample loading as described before ( 16 ).) w5 J: ?5 K, H, w* U+ v! @- m

Statistical analysis. Results are expressed as means ± SE of values obtained in triplicate from at least two different experiments. Differences between groups were compared using Student's t -test. P values / P, n3 B' `5 }. I0 P$ ~2 j

RESULTS
$ Q1 r9 b# _+ j) s  f
bFGF induces branching and proliferation of developing collecting ducts in cultured rat MK explants. To confirm that bFGF modulated the growth of developing tubules in our experimental system, E-15 MK explants were exposed to either 20 µl PBS (controls), rAd- lacZ (control vector), or rAd-bFGF, as described in MATERIALS AND METHODS ( n = 6 per group). As expected, rAd-bFGF induced the branching of developing tubules, as well as a remarkable duplication of DBA-positive cells underlying the collecting ducts ( Fig. 1 A ). These findings were reproduced using recombinant human (rh)-bFGF, and inhibited by bFGF-neutralizing antibodies ( Fig. 1 B ). It should be noted, however, that cultured rat MK exposed to rAd-bFGF or hrbFGF did not develop cysts in vitro. As anticipated, MK explants infected with rAd-bFGF showed a significant accumulation of bFGF ( Fig. 1 A, F ). In a similar manner, MK explants transduced with LacZ also expressed high levels of ( -gal) proteins (data not shown). No significant differences in the number of DBA-positive cells underlying branching tubular structures were found between the rat MK explants exposed to PBS (controls) or to rAd- lacZ control vectors ( Fig. 1 B ). Thus, based on these results, the MK transplant experiments were carried out using only rAd- lacZ vectors as controls.2 b% o0 a# ]( {* u, W# U- o$ |

Fig. 1. A : FGF-2 induces branching and proliferation of developing collecting ducts in cultured rat metanephric kidney (MK) explants. Top : representative low- and high-magnification pictures of rat MK explants transduced with rAd- Lac Z ( A ) or rAd-bFGF ( B ) and stained with Dolichos biflorus agglutinin (DBA). All MK explants were followed in vitro for 72 h, and the DBA-positive structures are shown in brown color (black arrows). C and D : representative immunohistochemical staining for the Phospho-p44/42 MAPK (Thr202/Tyr204), a potential signaling target of bFGF, in rat MK explants infected with rAd- Lac Z ( C ) or rAd-bFGF ( D; black arrows). E and F : immunohistochemical staining for bFGF in rat MK explants infected with rAd- LacZ ( E ) or rAd-bFGF ( F ). MK explants infected with rAd-bFGF show accumulation of bFGF mainly in the renal extracellular matrix surrounding developing tubules ( F ). Top scale bar = 300 µm. Bottom scale bar = 30 µm. B : graph represents changes in the number of DBA-positive cells underlying developing collecting ducts in each treatment group. Five different groups of cultured rat MK explants ( n = 6 per group) were exposed either to rA- Lac Z; rAd-bFGF; control buffer (20 µl PBS); recombinant human (rh)-bFGF (20 ng/ml); or rh-bFGF (20 ng/ml) plus a bFGF- neutralizing antibody (bFGF Ab), as described in MATERIALS AND METHODS. The number of DBA-positive cells underlying developing collecting ducts (black arrows) was quantified using computer-assisted image-analysis software. These results are expressed in arbitrary units (means ± SE), as described in MATERIALS AND METHODS. *,** P
; Z6 ]$ H: B. |5 P
rAd-bFGF induces renal cysts in rat MK explants transplanted under the renal capsule of adult rats. Next, we carried out experiments to determine whether the tubular proliferative changes induced by bFGF in vitro could induce the development of renal cysts in vivo. In these experiments, rat MK explants were transduced with either rAd-bFGF or rAd- lacZ ( n = 10 per group) and transplanted under the renal capsule of adult female Sprague-Dawley rats ( n = 6 rats per group). All rats were followed for up to 7-15 days. We found that 9 of 10 MK explants (90%) transduced with rAd-bFGF underwent a successful integration into the kidney of adult rats. In contrast, only 5 of 10 MK explants (50%) transduced with rAd- lacZ underwent a successful integration into the host kidneys. All MK explants transduced with rAd-bFGF developed tubular dilatation and microcysts, and eight of these MK explants developed large macroscopic cysts ( Fig. 2 A ). In contrast, only 2 of 10 MK explants transduced with rAd- lacZ developed small cysts, which were detected only under light microscopic examination. None of these MK explants developed macroscopic cysts during a similar follow-up period ( P 2 O$ ]0 p3 S: o& `+ Y. B7 [7 I: i

Fig. 2. Recombinant adenoviral vectors encoding the human basic fibroblast growth factor (bFGF) gene induced large macroscopic cysts in rat MK explants transplanted under the renal capsule of adult rats. A : representative macroscopic pictures of the rat MK explants infected with either rAd- LacZ (MK- LacZ ) or rAd-bFGF (MK-bFGF) and transplanted under the renal capsule of adult rats. The pictures were taken 15 days after transplantation. The white arrows point to the location of the transplanted MK explants. Scale bar: 0.5 cm. B - K : representative microscopic pictures of the rat MK explants successfully integrated into the renal cortex of adult rats. B and C : developing collecting duct cells stained red with DBA. D and E : similar sections at higher magnification. F and G : representative staining for proliferating cell nuclear antigen (PCNA; red). H and I : representative stainings for von Willebrand factor antigen (vWF; black arrows) in endothelial cells. J and K : representative stainings for the ED-1 antibody (red), which detects mononuclear cells infiltrating the transplanted MK explants. Scale bar: 25 µm ( B - K ). The graphs represent means ± SE corresponding to changes in the number of DBA-, PCAN-, vWF-, and ED-1-positive structures/cells expressed as % changes relative to the control group. Five MK explants were evaluated in each group (* P 8 B. E9 b9 h& X' e) I( I, [
/ K% h1 r  y+ W, j" }" [
FGF-2 induces renal cysts in newborn mice. To determine whether bFGF was capable of inducing the formation of renal cysts in developing mouse kidneys with an intact renal collecting system, we performed additional experiments in newborn mice. Here, we used an adenoviral-mediated renal transferring technique developed in our laboratory, which allows us to express foreign genes in vivo in developing mouse kidneys, as well as other tissues ( 12 ). By RT-PCR using specific human bFGF primers, we detected bFGF mRNA in the kidney of newborn mice infected with rAd-bFGF vectors, but not in adult mice infected in a similar manner with the same vector ( Fig. 3 ). The renal accumulation of human bFGF mRNA and protein was associated with the development of renal cysts and/or tubular dilatation in all newborn mice infected with rAd-bFGF ( Fig. 4 ). In contrast, none of the control mice injected with PBS (sham infections) or rAd- LacZ (control rAd vectors) developed tubular dilatation or renal cysts: sham infection group: 0.19 ± 0.3; rAd- lacZ control group: 0.21 ± 0.2 vs. rAd-bFGF group: 3.2 ± 0.8 arbitrary units (means ± SE) corresponding to the tubular dilatation scores, respectively ( P : w. ]: Q( R- A1 l9 E

Fig. 3. RT-PCR for human bFGF mRNA in the kidney of newborn mice injected with rAd-bFGF. Top lanes (bFGF) show RT-PCR amplification bands corresponding to human bFGF mRNA. Lanes 1 and 2 show renal samples corresponding to adult mice injected with rAd-bFGF. Lanes 3 and 4 show renal samples corresponding to newborn mice injected with rAd- lacZ. Lanes 5 and 6 show control renal samples from adult mice injected with rAd-bFGF but not subjected to the RT reaction. Lanes 7 and 8 show control renal samples from newborn mice injected with rAd-bFGF but not subjected to the RT reaction. Lane 9 shows an amplification band corresponding to the bFGF control plasmid. Bottom lanes (GAPDH) show RT-PCR amplification bands corresponding to mouse GAPDH in similar samples.! ^0 y2 `3 F" {' H: t

Fig. 4. Immunohistochemical staining for bFGF and phospho-p44/42 MAPK (p-ERK) in newborn mouse kidneys injected with rAd- lacZ and rAd-bFGF vectors. A : representative renal section from a newborn mouse injected with rAd- lacZ and stained with an affinity-purified IgG fragment (2.5 µg/ml) of a bFGF rabbit polyclonal antibody as described in MATERIALS AND METHODS. B : representative renal section harvested from newborn mice injected with rAd-bFGF and stained with the same bFGF antibody. C : representative renal section harvested from newborn mice injected with rAd-bFGF and stained with a control nonimmune IgG antibody (2.5 µg/ml). D : dilated renal tubular structures stained with the DBA (brown color) in the kidney of mouse infected with rAd-bFGF. E - H : representative immunohistochemistry pERK stainings in renal sections harvested from mice infected with rAd-bFGF. E : no specific p-ERK staining in a section incubated with the control non immune antibody. F - H : specific p-ERK staining in cystic epithelial cells, interstitial, and collecting duct cells (brown color, white arrows). Scale bars = 50 µm.

DISCUSSION. V$ \: z+ a* B: Q7 h6 s$ X

bFGF is a powerful angiogenic heparin-binding growth factor that is accumulated in the kidney and urine of HIV-infected children with renal microcysts secondary to HIVAN ( 16, 26 ). In the present study, we used two different adenoviral-mediated gene-transferring techniques to test the hypothesis that overexpression of bFGF induces the formation of renal cysts in developing rodent kidneys. We found that bFGF induced the formation of cysts in rat MK explants transplanted under the renal capsule of adult rats and in the kidney of newborn mice with an intact collecting system. To the best of our knowledge, these findings demonstrate for the first time that the accumulation of bFGF in developing rodent kidneys can induce the formation of renal cysts in vivo.

Previous studies have shown that bFGF plays important roles during renal development. Perantoni et al. ( 21 ) first showed that bFGF can induce the early events of renal tubulogenesis in isolated rat metanephrogenic mesenchymes. Subsequently, other investigators found that bFGF increases the survival and the expansion of stromal precursor cells in rat and mouse renal embryonic explants ( 1, 18 ). Taken together, all these studies support the notion that bFGF can modulate the process of renal development in rodents by inducing branching morphogenesis and proliferation of developing tubular epithelial cells, precursor mesenchymal cells, and renal capillaries ( 1 ). Because we used cultured E-15 MK explants undergoing many tubular branching events, we estimate the bFGF probably enhanced these branching events by inducing the proliferation of developing epithelial cells. However, we did not exclude the possibility that these changes could have been also mediated, at least partially, by stimulating the proliferation of mesenchymal cells. Our study also explored the in vivo role of bFGF during the process of renal development. To address this issue, we used a MK transplant model previously described by Hammerman and co-workers ( 27 ). These investigators showed that E-15 MK explants transplanted under the renal capsule of adult rats can develop small renal cysts 4-6 wk after transplantation. The process of spontaneous cyst formation in this experimental system is facilitated by the lack of connection between the ureter of the transplanted MK and the ureter or bladder of the host ( 15 ). This model, which develops what appear to be obstructive cysts, may be relevant for the pathogenesis of HIVAN, because the accumulation of proteinaceous material in the tubular lumen of these patients may also facilitate the obstruction of renal tubular structures. Thus, because bFGF is a powerful mitogen for renal tubular epithelial cells, it is possible that it could induce the formation of renal cysts through this mechanism. We found that bFGF induced the rapid formation of macroscopic renal cysts in less than 2 wk, in almost all MK explants transplanted under the renal capsule of adult rats. Subsequently, to determine whether bFGF was capable of inducing renal cysts in newborn mouse kidneys with an intact renal collecting duct system and no evidence of obstruction, we used an adenoviral-mediated renal gene-transferring technique for newborn mice developed in our laboratory ( 12 ). Considering that rodents, unlike humans, continue the process of nephrogenesis for the first 2 to 3 wk after birth, this technique allowed us to study the in vivo role of bFGF during renal development. Overall, our results confirmed that bFGF can induce the formation of renal cysts in developing rodent kidneys with an intact renal collecting system.

This study also demonstrates that a simple intravenous retroorbital injection of rAd-bFGF can induce a successful expression of human bFGF in the kidney, as well as other tissues of newborn mice ( 12 ). In previous studies, we developed two adenoviral-mediated gene-transferring methods named "portal-clamping" and "prolonged renal-infusion" techniques, to express genes in renal glomeruli of adult mice ( 38 ) and rats ( 39 ). Both methods are based on the principle of bypassing the hepatic circulation to prevent the rapid clearance of rAd from the circulation after the initial viral infusion. Both studies show that viruses need to be actively passing through the kidney for an extended period of time, at least for 15 min, to achieve efficient infection ( 38, 39 ). Most recently, we have shown that newborn mice have a delayed clearance of rAd vectors compared with adult mice ( 19 ). Thus it is tempting to speculate that the delayed clearance of rAd vectors from the circulation of newborn mice may increase the time of exposure of renal cells to circulating viruses and facilitate the infection of these cells. However, we did not rule out the possibility that other factors, like an upregulation of adenoviral receptors in newborn renal cells, and other hemodynamic or immunological developmental changes, may facilitate the infection of developing rodent renal cells. Alternatively, it should be noted that other tissues (i.e., liver) are also infected with this technique, and therefore bFGF released into the circulation by hepatocytes could also contribute to the accumulation of bFGF the kidney ( 24 ). Taken together, we have established two new adenoviral-mediated gene-transferring techniques to study the process of cyst formation in developing rodent kidneys.
1 e, f! V( C% B% h1 k. j
At the present time, we do not know the exact mechanisms by which bFGF induces the formation of renal cysts in developing rodent kidneys. Many cystic renal diseases are characterized by abnormal proliferation of tubular epithelial cells. Basic FGF can induce the proliferation of renal epithelial cells by activating extracellular signal-regulated kinases (ERKs), which are sensitive threonine kinases that regulate the expression of genes involved in cell proliferation and differentiation ( 29 ). In support of this notion, we have shown that bFGF induces the proliferation of primary human renal tubular epithelial cells by activating an ERK-dependent pathway ( 11 ). In a similar manner, activation of the ERK pathway induces the proliferation of renal epithelial cells harvested from patients with autosomal polycystic kidney disease ( 10, 34, 36, 37 ). Thus these studies, together with our p-ERK immunohistochemistry data, suggest that the cystogenic effect of bFGF in developing rodent kidneys may be mediated by an ERK-dependent pathway. In addition, at the present time, we do not know why bFGF causes cysts in vivo but not in vitro. We can only speculate that the cystogenic activity of bFGF may be regulated in a tissue-specific manner, depending on the type of heparan sulfate proteoglycans expressed on the cell surface and extracellular milieu ( 31 ). Thus it is likely that our tissue culture conditions are not reproducing the in vivo conditions. Alternatively, bFGF may stimulate a second cell type that is present in vivo but not in vitro (i.e, recruitment of circulating renal progenitor cells, endothelial, or inflammatory cells). Finally, we have shown that bFGF induces angiogenesis in vivo, and this process may induce hemodynamic changes that could favor the development of renal cysts. Further studies are needed to test all these hypothesis in developing rodent kidneys.+ k& \0 E# o4 I: s/ A

Of interest, another member of the FGF family, FGF-7 (keratinocyte growth factor), can also induce the formation of renal cysts. Transgenic mice expressing FGF-7 in the liver under the control of human ApoE gene promoter can develop a phenotype resembling the cystic changes in infants with polycystic kidney disease ( 20 ). Alternatively, FGF-7 null mice exhibit a reduced number of nephrons and smaller size of the renal collecting system compared with wild-type mice ( 23 ). FGF-7 is unique among other members of the FGF family, because it binds and signals through only one FGF receptor isoform (KGF), is produced exclusively by cells of mesenchymal origin, and only induces proliferation of epithelial cells ( 2, 6, 17 ). In contrast, bFGF does not signal through the KGF receptor isoform, is produced by almost all intrinsic renal cells, and induces the proliferation of many renal cell types ( 2, 6, 17, 31 ). Taken together, these data provide compelling evidence to support the notion that at least two different FGF receptor subtypes can regulate the cystogenic effects of FGFs. These findings could be relevant for the pathogenesis of pediatric renal diseases, because while the expression of FGF-7 is predominately restricted to embryonic kidneys ( 2, 6, 17, 30 ), bFGF is widely expressed throughout renal development, and in young and adult kidneys ( 4, 16, 19 ).0 b- R4 U) I+ U
0 g9 w* S2 {* S8 t. @" `! i: t
Basic FGF is a powerful angiogenic growth factor expressed among other tissues in endothelial cells ( 22 ). Our data also provide additional insights into the angiogenic role of bFGF during renal development. An interesting report by Kloth et al. ( 14 ) showed that bFGF induced the disappearance of vascular structures in cultured newborn rabbit kidney explants. In this study, the renal explants treated with bFGF were considerably thinner compared with the control kidneys, and the authors suggested that the accumulation of bFGF in dysplastic kidneys could paradoxically disrupt the normal growth of renal capillaries and impair the process of renal development. In contrast to these findings, Barasch et al. ( 1 ) reported that bFGF increased the survival of precursor endothelial cells in isolated cultured (E13) rat mesenchymes and demonstrated the ability of bFGF to rescue capillary precursors even at an early stage of the rat renal development process. Our studies support the notion that bFGF has a proangiogenic role during renal development. It is tempting to speculate that the angiogenic effects of bFGF may facilitate the rapid integration of the transplanted MK explants into the host kidneys. However, more studies are needed to prove this notion.

Our findings may be clinically relevant for developing human kidneys and HIV-infected children. In the human fetal kidney, bFGF is detected predominately in epithelial cells during different stages of differentiation ( 4 ). By in situ hybridization, bFGF mRNA can be detected in the ureteric bud epithelium, in newly formed "S-shaped bodies," in condensed "caps" of induced mesenchymal cells, and surrounding the extracellular matrix in early stages of cell condensation ( 4 ). This localization suggests that bFGF synthesized and released by ureteric bud cells can modulate the growth and survival of developing renal cells located nearby ( 1, 5, 18 ). Our results support and expand the findings of these studies and suggest that bFGF may participate in the process of cyst formation, at least partially, by inducing the proliferation of developing tubular epithelial cells. These findings also support the notion that bFGF may contribute to the development of renal microcysts in children with HIVAN. In agreement with this notion, bFGF is found in the urine of children with HIVAN ( 26 ), and primary renal tubular epithelial cells harvested from the urine of these children express high levels of bFGF and proliferate very rapidly ( 16, 26 ). In addition, we found that bFGF can increase the recruitment of circulating mononuclear cells in the kidney, and in a previous study we reported that bFGF increases the attachment of mononuclear cells to renal tubular epithelial cells harvested from children with HIVAN ( 35 ). Taken together, these findings suggest that bFGF may play a dual role in the pathogenesis of HIVAN, by increasing the recruitment of HIV-infected mononuclear cells and the formation of renal microcysts. In future studies, it will be important to determine how the spatial and temporal developmental pattern of expression of FGF's high- and low-affinity receptors may modulate the renal cystogenic activity of bFGF in HIV-infected kidneys.) |' D  }8 r0 Y* @. o1 r5 s* E

In conclusion, using two different adenoviral-mediated renal gene-transferring approaches, we demonstrated that the accumulation of bFGF in developing rodent kidneys can induce the formation of renal cysts in vivo. We are hopeful that these findings may provide a new mechanistic insight into the pathogenesis of childhood renal cystic diseases and facilitate the development of new approaches to treat, prevent, and/or monitor the progression of these renal diseases.) ]2 N! F$ z; U
& q+ e6 H3 p" A/ y
GRANTS
1 g& @& l: `8 `0 a6 x, M, `/ W0 q! ^
This work was supported by United States Public Health Service awards R0-1DK-4919 and RO-1HL-55605 and by (FADI) Buenos Aires, Argentina.* [0 s9 j2 w9 ?( T# N6 B( f5 m# {
      【参考文献】
Barasch J, Qiao J, McWilliams G, Chen D, Oliver JA, and Herzlinger D. Ureteric bud cells secrete multiple factors, including bFGF, which rescue renal progenitors from apoptosis. Am J Physiol Renal Physiol 273: F757-F767, 1997.

Celli G, LaRochelle WJ, Mackem S, Sharp R, and Merlino G. Soluble dominant-negative receptors uncover essential roles for fibroblast growth factors in multiorgan induction and patterning. EMBO J 17: 1642-1655, 1998.# {: L" s6 `5 ]+ h$ e

Clayton A, Thomas J, Thomas GJ, Davies M, and Steadman R. Cell surface heparan sulfate proteoglycan control the response of renal interstitial fibroblasts to fibroblast growth factor-2. Kidney Int 59: 2084-2094, 2001.

Drummond IA, Mukhopadhyay D, and Sukhatme VP. Expression of fetal kidney growth factors in a kidney tumor line: role of FGF-2 in kidney development and in dysplastic kidneys. Exp Nephrol 6: 522-533, 1998.; j  q; ]0 c3 o) g6 e" |
# D- W0 n2 ]; d- C
2 `' z5 U1 X1 B+ |# c$ u
( m" m4 [4 |2 \
Dudley AT, Godin RE, and Robertson EJ. Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme. Genes Dev 13: 1601-1613, 1999.7 j+ B& h# Y; S4 s
4 V! ^& }! s% J' I7 d. h
, w' r+ y8 t( B
9 }  N8 ^5 p$ V  c- O. w
Givol D and Yayon A. Complexity of FGF receptors: genetic basis for structural diversity and functional specificity. FASEB J 6: 3362-3369, 1992.1 G  K! ]- E# R9 j. O

5 \" y; l9 `$ p8 `0 x

Gonzalez AM, Hill DJ, Logan A, Maher PA, and Baird A. Distribution of basic fibroblast growth factor in the 18 day rat fetus. Localization in the basement membranes of diverse tissues. J Cell Biol 110: 753-765, 1990.
$ D, F6 Y+ E- r+ o

Gupta AR, Dejneka NS, D'Amato RJ, Yang Z, Syed N, Maguire AM, and Bennet J. Strain-dependent anterior segment neovascularization following intravitreal gene transfer of basic fibroblast growth factor (bFGF). J Gene Med 3: 252-259, 2001. <a href="/cgi/external_ref?access_num=10.1002/1521-2254(200105/06)3:3/ ]" E3 W7 Z7 r! k

Gupta GK, Milner L, Linshaw MA, McCauley RG, Connors S, Folkman J, and Bianchi DW. Urinary basic fibroblast growth factor: a noninvasive marker of progressive cystic renal disease in a child. Am J Med Genet 17: 132-135, 2000.
3 u* r0 z+ A/ ?

% g2 k$ b3 l/ r% r, _0 R
Hanaoka K and Guggino WB. cAMP regulates cells proliferation and cyst formation in autosomal polycystic kidney disease cells. J Am Soc Nephrol 11: 1179-1187, 2000.

7 z1 `3 l, q9 m6 |# X" [

Izevbigie EB, Gutkind JS, and Ray PE. Isoproterenol inhibits fibroblast growth factor-2 induced growth of renal epithelial cells. Pediatr Nephrol 14: 726-734, 2001.! R. ^2 I+ L; u. ^, ]% e9 s
$ U" X  o# Q5 X6 v( ~  Y9 ^
; O3 U3 e: Q) V" ?  Q; b. J; O

Jerebtsova M, Liu XH, Ye X, and Ray PE. Adenovirus-mediated gene transfer to glomerular cells in newborn mice. Pediatr Nephrol 20: 1395-1400, 2005.

) s, G7 K, o: V3 }- m6 E
, |/ \, ?2 a$ T7 ?$ Z
Jones SG, Morrisey K, and Phillips AO. Regulation of renal proximal tubular epithelial cell fibroblast growth factor-2 generation by heparin. Am J Kidney Dis 38: 597-609, 2001.

2 \7 B. w3 G/ e5 ]6 v: ?( {

Kloth S, Gerdes J, Wanke C, and Minuth WW. Basic fibroblast growth factor is a morphogenic modulator in kidney vessel development. Kidney Int 53: 970-978, 1998.

" B9 \2 D; |" ~7 I/ u. j' ]9 ~
' b$ L" W9 @  a5 P' k
Kozarsky K, Grossman M, and Wilson JM. Adenovirus-mediated correction of the genetic defects in hepatocytes from patients with familial hypercholesterolemia. Somat Cell Mol Genet 19: 449-458, 1993.+ `4 E- F0 R  z# L  e4 z9 K. O
/ I+ |. y! \' t# d) b. K5 f
9 t/ }7 ?1 n+ C, a6 U4 p) h

Liu XH, Achim A, Xu L, Wellstein A, and Ray PE. Upregulation of a fibroblast growth factor binding protein in children with renal diseases. Kidney Int 59: 1850-1858, 2001.
; g4 T- e& |. m% v* Z& B* d
( o# C7 e- z3 b# ?0 G" p5 ?/ `

Lovicu FJ and Overbeek PA. Overlapping effects of different members of the FGF family on lens fiber differentiation in transgenic mice. Development 125: 3365-3377, 1998./ ]/ q$ z3 R& B, G8 P
+ y4 _# z( f) l& {: r- j) h1 j# H8 Q
7 V' ^7 Z; f- O: l

Miyazaki Y, Oshima K, Fogo A, and Ichikawa I. Evidence that bone morphogenic protein 4 has multiple biological functions during kidney and urinary tract development. Kidney Int 63: 835-844, 2003." E" a9 V0 g+ l8 M  G& V

% e  j, L2 i9 l' \
" m& P! K/ o/ f: P& K
Morita H, Shizato T, David G, Mizutani A, Habuchi H, Fujita Y, Ito M, Asai Maeda KJ, and Kimata K. Basic fibroblast growth factor-binding domain of heparan sulfate in the human glomerulosclerosis and renal tubulointerstitial fibrosis. Lab Invest 71: 528-535, 1994.

Nguyen HQ, Danilenko DM, Bucay N, DeRose ML, Van GY, Thomason A, and Simonet WS. Expression of keratinocyte growth factor in embryonic liver of transgenic mice causes changes in epithelial growth and differentiation resulting in polycystic kidneys and other organs malformations. Oncogene 16: 2109-2119, 1996.
- q+ W: {2 L0 ?5 ^4 x
. f* o9 Z9 P  b0 P. q& y' E5 i

Perantoni AO, Dove LF, and Karavanova I. Basic fibroblast growth factor can mediate the early inductive events in renal development. Proc Natl Acad Sci USA 92: 4696-4700, 1995.

Powers CJ, McLeskey SW, and Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer 7: 165-197, 2000.
5 s% B# V( u/ E$ V) t
/ K3 R7 M* o6 V( @

Qiao J, Uzo R, Obara-Ishihara T, Degenstein L, and Fuchs E. FGF-7 modulates ureteric bud growth and nephron number in the developing kidney. Development 126: 547-554, 1999.7 C: M8 Y! U* a) V2 T8 ]& f$ x# r

Ray Bruggeman L PE, Weeks B, Kopp J, Bryant J, Owens J, Notkins A, and Klotman PE. Role of bFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice. Kidney Int 46: 759-772, 1994.. E3 y7 d: Q3 T6 M8 z& g
/ F! a+ v/ z& k( b
9 m, l: t# v9 ], X' B
9 [; a  A2 v, B: i$ z
Ray PE, Liu XH, Robinson RL, Reid W, Xu L, Owens JW, Jones OD, Denaro F, Davis HG, and Bryant JL. A novel HIV-1 transgenic rat model of childhood HIV-1 associated nephropathy. Kidney Int 63: 2242-2253, 2003.4 i" h0 m$ A8 @4 i: M
7 D  _6 @. O0 i3 t+ S  e9 V

" h4 l  |# L) |. X% [% _/ c
Ray PE, Liu XH, Xu L, and Rakusan T. Accumulation of bFGF in children with HIV-associated hemolytic uremic syndrome. Pediatr Nephrol 13: 586-593, 1999.% `( t5 Y; k: I! f. ?

Rogers SA, Lowell JA, Hammerman NA, and Hammerman MR. Transplantation of developing metanephroi into adult rats. Kidney Int 54: 27-37, 1998.3 I0 @  s9 B0 s3 F7 |! E+ ]
& B9 i1 ^( V9 P* ~' h/ n' b

Rogers SA, Ryan G, and Hammerman MR. Insulin-like growth factors I and II are produced in the metanephros and are required for growth and development in vitro. J Cell Biol 113: 1447-1453, 1991.
. q+ f' c: o& f! U- e: X( E
. P4 Q/ i' B9 c, r8 }

Rozengurt E. Early signals in the mitogenic response. Science 234: 161-166, 1986.! E& F+ c( v$ W3 M

, q8 E3 n9 p( q+ E, n, e
Shima Tazawa H and Puri P. Increased expression of fibroblast growth factors in segmental renal dysplasia. Pediatr Surg Int 16: 306-309, 2000.+ r. j* c) s3 i# p$ d

/ n  K+ d7 W' g& D
; p! V# L. C1 w; ]
Steer DL, Shah MM, Bush KT, Stuart RO, Sampogna RV, Meyer TN, Schwesinger C, Bai X, Esko JD, and Nigam SK. Regulation of ureteric bud branching morphogenesis by sulfated proteoglycans in the developing kidney. Dev Biol 272: 310-327, 2004.0 z: W# o8 k8 V) R0 T
% }; Z) [, y7 y! x! J8 C

) c* F- ~3 s5 S: _  w" C) ]
Strutz F, Zeisberg M, Hemmerlein B, Sattler B, Hummel K, Becker V, and Muller GA. Basic fibroblast growth factor expression is increased in human renal fibrogenesis and may mediate autocrine fibroblast proliferation. Kidney Int 57: 1521-1538, 2000.  ^0 ]" C0 n' F3 i( R4 o
- ~7 O' E, H9 ~; C' h* M

Struzt F, Zeisberg M, Ziyadeh FN, Yang CQ, Kalluri R, Muller GA, and Neilson EG, with the technical assistance of Renziehausen A and Sisic Z. Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation. Kidney Int 61: 1714-1728, 2002.
+ q4 T% t+ S" a  v& `0 @- h9 I9 j

- u: Y4 z, w. @' H( I5 d
Sutters M, Yamaguchi T, Maser RL, Magenheimer BS, St John PL, Abrahamson DR, Grantham JJ, and Calvet JP. Polycystin-1 transforms the cAMP growth-responsive phenotype of M-1 cells. Kidney Int 60: 484-494, 2001.. q( z( D+ `0 t4 ?, R: @
" I  j9 O! Y1 r3 z" g" @

Tang P, Jerebtsova M, Liu XH, and Ray PE. FGF induces the attachment and renal recruitment of HIV-infected mononuclear to renal tubular epithelial cells. Pediatr Nephrol 20: 1708-1716, 2005.

Yamaguchi T, Pelling JA, Ramaswamy NT, Eppler JW, Wallace DP, Nagao S, Rome LA, Sullivan LP, and Grantham JJ. cAMP stimulates the in vitro proliferation of renal cysts epithelial cells by activating the extracellular signal-regulated kinase pathway. Kidney Int 57: 1460-1471, 2000.
/ \5 s( g5 g* ?! g- j
- S, X9 A6 k4 M, l* K5 m; d
+ J- d& E& K" {; L) J2 ^
Yamaguchi T, Wallace DP, Magenheimer BS, Hempson SJ, Grantham JJ, and Calvet JP. Calcium restriction allows cAMP activation of the B-Raf/ERK pathway, switching cells to a cAMP-dependent growth stimulated phenotype. J Biol Chem 279: 40419-40430, 2004.
! U* I, X+ ~+ b# s. \& b1 M

' v$ r7 |1 D) j$ H2 ~) ]6 t, \0 v+ [
Ye X, Jerebtsova M, and Ray PE. Liver bypass significantly increases the transduction efficiency of recombinant adenoviral vectors in the lung, intestine and kidney. Hum Gene Ther 11: 621-627, 2000.# @  r' S( J+ X/ }8 `

1 v, I4 @4 B- z
7 ]9 g8 j5 M2 A5 t6 q3 N. w
Ye X, Liu X, Li Z, and Ray PE. Efficient gene transfer to rat renal glomeruli with recombinant adenoviral vectors. Hum Gene Ther 12: 141-148, 2001.

收藏0 分享0 顶0 踩0