Radiation exposure to the kidney causes acute and chronic injuries that could eventually lead to the development of fibrosis and end-stage organ failure.1 Such injury is a dose-limiting factor for irradiation therapy. Although studies on the role of acute irradiation on the kidneys are available, the chronic effects of such irradiation in kidneys are not studied in similar depth and details. For instance, the particular cell types and mediators involved in radiation-induced renal fibrosis are still not clearly defined. Following radiation exposure, renal cells may transdifferentiate into myofibroblasts by a conversion known as tubular epithelial-mesenchymal transition (EMT),2 and might contribute to the development of tubulointerstitial fibrosis.
Connective tissue growth factor (CTGF) has recently received much attention as a novel profibrotic factor, it has been identified as an important mediator of fibrosis maintenance in various pathological conditions, including radiation enteritis.3 In this study, we have monitored the role of CTGF in radiation nephropathy, determined the expression of CTGF by immunohistochemistry and identified the CTGF-expressing cells by double immunostaining in the irradiated kidney. More importantly, we have found that there is a strong association between the expression of CTGF and the progression of radiation nephropathy. The results suggest that the expression of CTGF is closely associated with phenotypic transformation of renal cells into myofibroblasts, which might contribute to excessive synthesis of collagens to induce renal fibrosis in radiation nephropathy. Increased expression of CTGF, therefore, may play an important role in the development of glomerulosclerosis and tubulointerstitial fibrosis in radiation nephropathy.
Male Wistar rats (n=32), aged 6 weeks, were divided into two experimental groups. Radiation procedures were previously reported.4 Briefly, control group (n=12) included age-matched control rats received only laparotomy without irradiation. For rats in the irradiated group (n=20), after intraperitoneal nembutal anesthesia (25 mg/kg of body weigh), the kidneys were exposed through the surgical incision and covered with sterile gauze saturated with physiologic saline solution. The exposed kidneys were given a single-dose of 25 Gy X-ray irradiation through a specially designed lead shield, which protected the entire body and only allowed irradiation of the kidneys.
Renal function studies
The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were measured by autoanalyzer (Hitachi 7170, Hitachi City, Japan).
At each day of the first, third, sixth and ninth month after radiation, three rats of the control group and five rats of the irradiated group were examined. The kidneys were rapidly removed and weighed and fixed immediately in 10% formalin for 24 hours and processed further for histological and immunohistochemical examination.
Tissue were processed and embedded in paraffin, cut into 4-μm sections and stained with hematoxylin-eosin (HE), periodic acid-Schiif (PAS), periodic acid-methenamine sliver (PAM) and Masson trichrome. The extent of glomerular, tubulointerstitial and vascular damage was determined by light microscopy.2,4 Glomeruli were scored individually based on the averaged percentage of sclerotic area of the glomerular tuft as follows: 0, no sclerosis; 1, sclerotic area <5%; 2, sclerotic area 5%-24%; 3, sclerotic area 25%-50%; 4, sclerotic area ≥50%. Tubulointerstitial injuries were semiquantified using PAS-stained sections and were scored as follows: 0, no injury; 1, injury involved <5%; 2, injury involved 5%-24%; 3, injury involved 25%-50%; 4, injury involved ≥50%.
The detailed immunohistochemical procedure was described earlier.4 Briefly, paraffin sections (4 μm) were deparaffinized with xylene, rinsed thoroughly with ethanol, then soaked in 0.03% hydrogen peroxide in methanol to inactivate endogenous peroxidase activity. The sections were incubated with either 10% goat serum or 10% rabbit serum, then covered with primary antibodies, washed with phosphate-buffered saline (PBS) and processed further using Histofine SAB-PO Kit (Nichirei, Tokyo, Japan), as directed by the manufacturer, and developed with 3, 3′-diaminobenzindine (DAB) and H2O2. Primary antibodies against the following antigens were used: CTGF (Abcam, Cambridge, UK), α-SMA (Dako, Glostrup, Denmark), vimentin (Dako), type III collagen (Chemicon, Temecula, CA, USA), type IV collagen (Chemicon). The results were calculated as the percentage of positive-stained area and the total field area then expressed as graded data to do correlation analysis. The staining intensity in glomeruli and in tubulointerstitium was graded according to the following scales: 0, negative; 1, a trace (<5%); 2, mild (5%-24%); 3, moderate (25%-50%); 4, severe (≥50%). A 100-point eyepiece grid was used for point counting (Zeiss, Welsyn Garden City, UK). Scores were calculated for at least 30 eye-fields at magnification ×400, summed and averaged for each time point.
Double immunostaining was performed to define co-localization of CTGF with α-SMA, vimentin, type III collagen and type IV collagen in the same renal section, as described earlier.5 Sections were processed as above except that the sections were incubated first with the polyclonal antibody against CTGF for 1 hour, then with biotinylated second antibody and streptavidin-alkaline phosphatase, and developed with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT), which produced a dark purple stain. The CTGF-stained slides were then counterstained with α-SMA, vimentin or collagens by the streptavidin-biotin-peroxidase method and the antigen-antibody complex was visualized by aminoethyl carbazole (AEC)/H2O2, which produced an intense red stain.
Western blotting analysis
The renal cortical tissues were removed and frozen immediately; the frozen tissues were then suspended in lysis buffer, supernatants were collected and the protein concentration was measured with an ultraviolet/visible spectrophotometer (UV-1600; Shimadzu, Japan). Equal amounts of protein samples (50 μg/lane) were subjected to electrophoresis on 12% SDS-PAGE gel and transferred to PVDF membrane (Amersham Life Science, USA). The membranes were blocked with 5% nonfat dry milk in TBST buffer. Immunoblot analyses was performed with rabbit polyclonal anti-CTGF (Abcam, Cambridge, UK). The membranes were incubated overnight at 4°C with primary antibodies, washed in TBST buffer and further incubated with sheep anti-mouse IgG or donkey anti-rabbit IgG coupled to horseradish peroxidase in TBST buffer for one hour by using the RPN 2108 ECL Western blotting analysis system (Amersham Pharmacia Biotech, USA). Immunoreactive protein was detected by enhanced chemiluminescene, according to the manufacturer's instructions. The ECL detected blots were exposed to Polaroid film using the ECL mini-camera.
Statistical analysis was carried out using StatView-J 5.0. The data are shown as a mean ± standard deviation (SD). Statistical comparisons were executed by one-way analysis of variance (ANOVA), chi-square test and Student's t test depending on different types of data. Correlation analysis was performed by Spearman's rank correlation analysis. Statistical significance was achieved at corrected P <0.05.
The level of BUN and SCr in the irradiated group began to markedly increase from the first month after irradiation (P <0.01, Table 1). Compared to the control group, the level of BUN and SCr were significantly higher at the third, sixth and ninth month after irradiation (P <0.001, Table 1).
Table 2 details the light microscopic morphological changes of kidneys at different times in the various groups. No significant histological changes were noted in the control kidneys in the whole experimental period (Figure 1A). In irradiated rats, glomeruli showed focal segmental sclerotic lesions at an early stage (Figure 1B).
The glomerular lesion gradually developed global glomerulosclerosis over time (Figures 1C and 1D). The damage to tubular epithelium was composed of cellular degeneration, atrophy, dilatation and thickening of tubular basement membrane, tubular hyalinous casts were also noted in places in the irradiated rat kidneys, the interstitium showed chronic inflammatory cell infiltration and fibrosis (Figure 1C), and became wider and more severer during the progression of the experimental period (Figure 1D). Radiation-induced glomerular and tubulointerstitial injuries were particularly severe the sixth month after irradiation as compared to the control group (P <0.01, Table 2), and remained severe when examined nine month after irradiation (P <0.001, Table 2).
Expression of CTGF
The patterns of immunochemical staining in different components of the kidney are summarized in Table 3. By immunohistochemistry, CTGF was weakly detected in the glomerular cells and interstitial cells in control rat kidneys (Figure 2A). In contrast, markedly increased immunostaining of CTGF was noted in the interstitial cells and in the tubular epithelial cells, interstitial fibrotic regions and glomerulosclerosis during radiation nephropathy (Figure 2B). Expression of CTGF began to increase from the first month after irradiation as compared to the control group (P <0.05, Table 3), and remained significantly higher at the sixth and ninth month after irradiation (P <0.01, Table 3).
Expression of type III collagen
Type III collagen was weakly stained in the control rat kidneys (Figure 3A). Markedly increased expression of type III collagen was noted in widen interstitial fibrosis during radiation nephropathy (Figure 3B). Compared to control rat kidneys, increased deposition of type III collagen was noted at the first and third month after irradiation (P <0.05, Table 3), and remained high at the sixth and ninth month after irradiation (P <0.01, Table 3).
Expression of type IV collagen
Control kidneys showed mild but distinct expression of type IV collagen in glomerular basement membrane and tubular basement membrane in irradiated rat kidneys (Figure 4A). In comparison to the control rat kidneys, increased deposition of type IV collagen was noted in the region of glomerulosclerosis and thickened tubular basement membrane (Figure 4B). Compared to control rat kidneys, increased accumulation of type IV collagen was noted at the first and third month after irradiation (P <0.05, Table 3), and remained high at the sixth and ninth month after irradiation (P <0.01, Table 3).
Expression of α-SMA
Alpha-SMA was present mainly in the vascular wall in the control rat kidneys (Figure 5A). An increased number of glomerular and interstitial cells expressed α-SMA in kidneys obtained from irradiated rats (Figure 5B). Expression of α-SMA began to markedly increase from the first and third months after irradiation as compared to control rat kidneys (P <0.05, Table 3), and remained significantly increased at the sixth and ninth month after irradiation (P <0.01, Table 3).
Expression of vimentin
In the control rat kidneys vimentin was weakly positive in the glomeruli, but negative in tubular epithelial cells (Figure 6A). In radiation nephropathy tubular epithelial cells and interstitial cells in and around the fibrous areas showed strong immunostaining for vimentin (Figure 6B), and was parallel to the progression of tubulointerstitial fibrosis associated with radiation nephropathy. Expression of vimentin began to markedly increase from the first and third month after irradiation as compared to control rat kidneys (P <0.05, Table 3), and significantly increased at the sixth and ninth month after irradiation (P <0.01, Table 3).
Co-localization of CTGF and type III, IV collagen
The CTGF-expressing cells were mostly found in and around increased deposition of type III collagen, particularly in the areas of interstitial fibrosis in radiation nephropathy (Figure 7A). Co-expression of both CTGF and type IV collagen was noted around the thickened TBM and interstitial fibrosis (Figure 7B).
Double immunostaining CTGF and α-SMA, vimentin
CTGF was expressed in α-SMA-positive cells in interstitial fibroblast-like cells in and around the tubulointerstitial fibrotic areas (Figure 7C). In glomeruli with proliferation and sclerosis, CTGF was overexpressed in association with increased expression of α-SMA (Figure 7C). By double staining, expression of CTGF was frequently noted in the tubular epithelial cells positive for vimentin in damaged fibrotic areas (Figure 7D).
Western blotting for CTGF
The 38 kDa CTGF protein expression markedly increased in radiation treated kidneys compared with the control rat kidneys (Figure 8); CTGF protein level in the irradiated kidneys began to increase at the first month and remained elevated at six and nine months after irradiation, which is in accord with the results of the immunohistochemistry.
The relationship between the expression of CTGF, type III collagen, type IV collagen, α-SMA and vimentin with the degree of renal injuries after irradiation is presented in Table 4. Analysis by linear regression showed that glomerular expression of CTGF positively correlated with the expression of α-SMA (r=0.628, P <0.01), vimentin (r=0.462, P <0.05), deposition of type IV collagen (r=0.584, P <0.01), but there was no significant correlation with deposition of type III collagen (r=0.326, P <0.05) in the irradiated glomeruli. Furthermore, CTGF expression in the tubulointerstitium appeared to be positively correlated with the expression of α-SMA (r=0.613, P <0.01), vimentin (r=0.629, P <0.01), deposition of type III collagen (r=0.741, P <0.001) and type IV collagen (r=0.799, P <0.0001). Furthermore, the glomerular and interstitial expression of CTGF in the irradiated kidney was strongly correlated with increased levels of BUN (r=0.726, P <0.001; r=0.656, P <0.001, respectively), and SCr (r=0.671, P <0.01; r=0.596, P <0.01, respectively).
Radiation nephropathy has emerged as a significant complication of bone marrow transplantation and radionuclide radiotherapy and is a potential sequela of radiological terrorism and radiation accidents.6,7 The pronounced radiosensitivity of renal tissue sometimes limits the use of radiotherapeutic treatment when required.8 With more and more radioactive resources introduced to our daily life and medical interventions, there is a great demand for understanding the detailed mechanism of radiation induced injury and searching for possible preventive targets. In our long-term study, local irradiation to the kidney of the experimental rats showed glomerulosclerosis and tubulointerstitial injuries, such as inflammatory infiltrates, tubular atrophy, degeneration and interstitial fibrosis. The renal lesions developed gradually throughout the experimental time course. Renal dysfunction due to radiation exposure was a time-dependent process. We have reported that the renal lesions occurred after a single exposure to X-ray irradiation (7–25 Gy) and appeared to be dose-dependent.4,9 As the mediators of tubulointerstitial fibrosis in radiation nephropathy have received scant attention, we emphasize the importance of recognizing the development of tubulointerstitial fibrosis in radiation nephropathy, as well as glomerulosclerosis.
CTGF is a 38 kDa cysteine-rich, secreted protein belonging to the CCN family of matricellular proteins.10,11 It is involved in matrix remodeling during controlled healing and uncontrolled tissue scarring and has increasingly been recognized as an important pro-fibrotic factor. EMT is a complex process in which renal tubular cells lose their polarized tubular epithelial phenotype and acquire new features characteristic of a mesenchymial phenotype, the major effector cells responsible for the excessive matrix deposition under pathologic conditions. CTGF has been reported to induce EMT directly and indirectly both in vivo and in vitro in various models.12–14
The main finding of this study was that CTGF was strongly expressed in tubulointerstitial fibrosis and glomerulosclerosis in irradiated rat kidneys as detected by immunohistochemistry. Overexpression of CTGF was closely associated with increased deposition of collagens type III and IV. Double immunostaining revealed co-localization of CTGF and collagens in the sclerotic/fibrotic areas. CTGF-expressing cells might be found to be phenotypically altered cells such as vimentin-positive tubular epithelial cells and α-SMA-positive interstitial myofibroblasts. Double immunohistochemistry staining procedure showed that the phenotypic alteration of tubulointerstitial cells was responsible for the increased expression of CTGF in irradiated rat kidneys. Increased expression of CTGF/type III collagen and CTGF/type IV collagen was parallel to the histopathological changes in the irradiated kidney. Upon Western blot analysis, CTGF protein expression began to increase after irradiation and appeared progressively throughout time course. These results suggested that CTGF may play a curial role in the transdifferentiation process of tubular epithelial cells in radiation nephropathy.
Using a rat model of radiation nephropathy, we have investigated the role of α-SMA at different time points and the relations with other cell phenotype, in order to make further steps in exploring the relations between tubular EMT and interstitial fibrosis.15 Although α-SMA is classically a major marker of EMT, vimentin has emerged as another important marker of EMT; considering EMT would perform like a two-steps processes, (1) epithelium to fibroblast then (2) fibroblast to myofibroblast. this new model in addition to the traditional conception of directly transforming from epithelium to myofibroblast.16 Recently, we observed that co-localization of α-SMA and vimentin in tubular epithelial cells in irradiated kidneys that suggested that EMT of tubular epithelial cells after radiation might include the above two processes.15 In the present study, we employed both of the two important markers for the purpose of avoiding any possible underestimation of EMT.
Factors regulating increased synthesis of CTGF in rat radiation nephropathy are not clear from this study. TGF-β1 has been described as the main CTGF inducer.17 A fibrogenic role of TGF-β1 was reported in radiation-induced nephropathy.18,19 TGF-β1 and its downstream effector CTGF, are known fibrogenic activators, and were found to be regulated by the Rho/ROCK pathway, and perhaps control CTGF expression in intestinal smooth muscle cells isolated from patients with delayed radiation enteritis.20 CTGF expression is coordinately upregulated with TGF-β1 in proliferative and fibrotic glomerular and interstitial lesions of many kinds of human and experimental nephropathies, and related closely with the degree of renal tubulointerstitial fibrosis. Studies of Zhang et al21 indicate that CTGF could promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblasts in vitro, both directly and as a downstream mediator of TGF-beta, and CTGF blockade would be a possible therapeutic target against tubulointerstitial fibrosis. Thus, based on the results of earlier studies, we speculate that increased expression of CTGF in irradiated kidneys might be induced by TGF-β1. As the biological actions of TGF-beta are complex and affect many different cell types, CTGF may serve as a more specific target for selective intervention in various fibrotic disorders.22–26 Therefore, selective inhibition of CTGF bioactivity by a neutralizing antibody might be a more specific anti-fibrotic target, particularly relevant in radiation-induced renal fibrosis.
From the results of our study, it is assumed that up-regulation of CTGF in radiation nephropathy may contribute to glomerulosclerosis and tubulointerstitial injury, possibly by promoting the transdifferentiation of renal tubular epithelial cells towards myofibroblasts and regulating increased production of collagens. Tubular EMT plays a key role in the initiation and development of tubulointerstitial fibrosis in radiation nephropathy. However, the mechanism regulating tubular EMT process remains largely unknown. Additional studies are needed to understand fully the respective roles of CTGF in radiation nephropathy to improve the therapeutic treatment of renal tubulointerstitial fibrosis.
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