Role of α-smooth muscle actin on radiation nephropathy in rats: Possibly an early marker of determination of fibrosis
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《中华医药杂志》英文版
Role of α-smooth muscle actin on radiation nephropathy in rats: Possibly an early marker of determination of fibrosis (pdf)
Department of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, Jiangsu Province 210009, China
Correspondence to LIU Diange, MD, PhD,Department of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Dingjiaqiao 87, Nanjing, Jiangsu Province 210009, China
Tel: +86-25-83285139，Email: liudiange2003＠yahoo.com. cn
[Abstract] Objective The molecular mechanisms of fibrosis in radiation nephropathy have received scant attention. However, the precise mechanism of tubulointerstitial injury in this condition is still unclear. Glomerular and tubulointerstitial α-smooth muscle actin (α-SMA) have been associated with progressive glomerulosclerosis and tubulointerstitial fibrosis. We observed expression and significance of α-SMA on the progression of radiation nephropathy in rats. Methods The model of radiation nephropathy with rats were established as follows: control group (n=12)；irradiated group (n=20), after right nephrectomy, a single dose 25 Gy Xray to left kidney. The rats were followed up for 1, 3, 6 and 9 months after renal exposure to radiation. The kidneys were examined by immunohistochemical method including double immunostaining.
Results Renal dysfunction was noted early in irradiated rats, along with inflammatory cell infiltration and interstitial fibrosis. Compared to control rat kidneys, increased expression of α-SMA and vimentin were noted in the early stage in irradiated kidneys. Further, expression of α-SMA was carrying on increase along with the progression of radiation nephropathy. Double immunostaining demonstrated tubular epithelial cell with vimentinpositive expression coexisted expression of α-SMA. Moreover, α-SMApositive myofibroblasts in the interstitium also appeared vimentinpositive expression. These findings showed that there were abundant cells coexpressing both α-SMA and tubular marker, indicating that they are at transitional stage between epithelial and mesenchyme. Overexpression of α-SMA and vimentin were closely associated with increased deposition of type Ⅲ and Ⅳ collagens in the widened interstitium of irradiated rats. Conclusions The phenotype change tubular epithelial cell possibly induced renal resident cell to occur the transdifferentiation, and participated in the development of fibrotic event in radiation nephropathy. The α-SMA might be regarded as index of early prognosis and determination of tubulointerstitial fibrosis in radiation nephropathy.
[Key words] α-smooth muscle actin; vimentin; mmunohistochemistry; radiation nephropathy;tubulointerstitial fibrosis
INTRODUCTION
Sufficient irradiation on the kidney causes progressive injury results in fibrotic process and organ failure. This injury is an example of normal tissue radiation injury, which is a doselimiting problem for all therapeutic irradiation. More studies have focused on a primary role for glomerular cells and tubular epithelial cells in radiationinduced renal injury. Especially in chronic phase of radiation nephropathy, renal fibrosis resulted from increased accumulation of various extracellular matrices (ECM) is a prominent feature. However, the particular cell types, mediators and mechanisms involved are still ill defined. Activation α-SMApositive myofibroblasts is considered a key event in the progression of renal fibrosis. Under pathologic conditions, tubular epithelial cells may transdifferentiate into myofibroblastsby a conversion known as tubular epithelialmesenchymal transition (EMT) [1], and participate in the development of tubulointerstitial fibrosis. We have reported that role of heat shock protein 47 (HSP47) in experimental radiation nephropathy, upregulation of HSP47 in phenotypically altered renal cells might contribute to synthesis of collagens resulting in tubulointerstitial fibrosis in radiation nephropathy [2]. The present studies used a rat model of radiation nephropathy to investigate expression and significance of α-SMA on the progression of radiation nephropathy. This study was focused on colocalization of vimentin and α-SMA by double immunostaining method, in order to elucidate the role of α-SMA on the progression of radiation nephropathy.
The results suggest that phenotypically altered renal cells might contribute to synthesis of collagens resulting in tubulointerstitial fibrosis in radiation nephropathy. The α-SMA might be regarded as an early marker of determination of tubulointerstitial fibrosis in radiation nephropathy.
METHODS
Experimental AnimalsMale Wistar rats (n=32), aged 6 weeks, were divided into two experimental groups. Radiation procedures were previously reported [2]. Briefly, control group (n=12) included agematched control rats received only laparotomy without irradiation. For rats in irradiated group (n=20), after intraperitoneal nembutal anesthesia (25 mg/kg of body weight), were nephrectomied for their right kidneys before radiation procedures. The left kidney was exposed through the surgical incision and covered with sterile gauze saturated with physiologic saline solution. The exposed left kidney was a singledose 25 Gy Xray irradiated through a specially designed lead shield, which protected the entire body and only allowed irradiation of left kidney. The irradiation source was provided by a 200 kV, 15 mV, superficial therapy unit (filter 0.05Al, Xray machine, Toshiba, Tokyo, Japan)，through the distance from target to organ measuring 50 cm. After treatment, the kidney was restored to its original position, and animals were given a regular diet and water. All the experimental procedures followed the guideline for care and use of laboratory animals.
Renal Function StudiesBlood was collected from the superior vena cava from different group of rats, at various time points, when the rats were killed. The levels of blood urea nitrogen (BUN) and serum creatinine (Cr) were measured by autoanalyzer (Hitachi 7170, Hitachi City, Japan).
Tissue CollectionAt each day of the first, third, sixth and ninth month after radiation, three rats of control group and five rats of irradiated group were examined respectively. The kidneys were rapidly removed and weighed, the fixed immediately in 10% formalin for 24 h and processed further for histological and immunohistochemical examination.
Histological StudiesTissue were processed and embedded in paraffin, cut into 4 μm sections and stained with hematoxylineosin (HE), periodic acidSchiff (PAS), periodic acidmethenamine sliver (PAM) and Masson trichrome. The extent of glomerular, tubulointerstitial and vascular damages was examined by light microscopy[2]. Glomeruli were scored individually based on the averaged percentage of sclerotic area of glomerular tuft as follows: -, no sclerosis; ±, sclerotic area <5%; +, sclerotic area 5%~25%; ++, sclerotic area 25%~50%; +++, sclerotic area >50%. Tubulointerstitial injuries were semiquantified using PASstained sections and were scored as follows: -, no injury; ±, injury involved <5%; +, injury involved 5%~25%; ++, injury involved 25%~50%; +++, injury involved >50%. The vascular wall thickening and luminal narrowing as follows: -, no thickening; +, mild thickening; ++, moderate thickening; +++, severe thickening.
ImmunohistochemistryImmunohistochemical methods were performed as described elsewhere [2~4]. Briefly, paraffin sections (4 μm) were deparaffinized with xylene, rinsed thoroughly with ethanol, then soaked in 0.3% hydrogen peroxide in methanol for 30 min at room temperature to inactivate endogenous peroxidase activity. After mild treatment with 0.05% trypsin (T 4799, Sigma, St Luis, MO, USA) for 5 min, the sections were incubated with either 10% goat serum or 10% rabbit serum for 30 min, then covered with primary antibodies, washed with phosphatebuffered saline (PBS) and processed further using Histofine SABPO 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: α-SMA (Dako, Glostrup, Denmark), vimentin (Dako), type Ⅲ collagen (Chemicon, Temecula, CA, USA), type Ⅳ collagen (Chemicon). The staining intensity was graded semiquantitatively according to the following scales [2]: -, no staining; ±, staining area <5%; +, staining area 5%~25%; ++, staining area 25%~50%; +++, staining area >50%. The extent of α-SMA immunostaining was scored as previously reported [1]: 0, negative; 1, traced (<5%)；2, mild (5%~25%)；3, moderate (25%~50%)；4, severe (>50%). For each group of slides, five adjacent eyepiece grid areas were studied, starting just below the renal capsule, and proceeding toward the medulla. A 100point eyepiece grid was used for point counting (Zeiss, Welsyn Garden City, UK). Scores were calculated for at least 30 eyefields at magnification 400, summed, and averaged for each time point.
Double ImmunostainingDouble immunostaining for vimentin and α-SMA was performed as described earlier [2~4]. Sections were processed as above except that the sections were incubated first with the monoclonal antibody against vimentin for 1 h, then with biotinylated second antibody for 10 min and streptavidinalkaline phosphatase for 10 min, and developed with 5bromo-4chloro3indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT), which produced a dark purple stain. Then the vimentinstained slides were counterstained with α-SMA by the streptavidinbiotinperoxidase method and antigenantibody complex was visualized by aminoethyl carbazole (AEC)/ H2O2, which produced an intense red stain. In between the first and second staining, the sections were autoclaved in citrate buffer (pH 6.0) for 15 min to minimize the cross reactivity between the first and second staining. As immunohistochemical control, primary antibodies were replaced with either 0.01 M PBS or mouse IgG/rabbit IgG diluted with PBS in a concentration similar to that of the primary antibodies.
Statistical Analysis
Statistical analysis was carried out using StatViewJ5.0. The data are shown as a mean ± SD. Comparisons between time points were made by first testing for group differences by Students ttest. Statistical significance was achieved at corrected P<005.
RESULTS
Blood ChemistryThe level of BUN and Cr in irradiated group began to markedly increase from the first, third month after irradiation (BUN: 27.7±0.31 vs 16.5±0.20 mg/dl, 44.3±0.38 vs 16.8±0.32; Cr: 0.49±0.08 vs 021±0.07 mg/dl, 0.62±0.08 vs 0.23±0.06 mg/dl; P<0.05, respectively). Compared to the control group, the level of BUN and Cr were significantly raised at the sixth and ninth month after irradiation (BUN: 98.5±0.40 vs 17.9±0.26 mg/dl, 192.3±4.78 vs 18.5±0.20 mg/dl; Cr: 1.42±0.07 vs 0.26±0.09 mg/dl, 2.68±0.08 vs 0.31±0.08 mg/dl; P<0.001, respectively). The renal function was parallel to the histological behaviors.
MorphologyTable 1 details the light microscopic morphological changes of kidneys at different times in 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 lesion at early stage (Figure 1b). The glomerular lesion gradually developed global glomerulosclerosis in time course (Figure 1c, d). The damages of tubular epithelium were composed of cellular degeneration, atrophy, dilatation and thickening of tubular basement membrane, tubular hyalinous casts were also noted in places in irradiated rat kidneys, the interstitium showed chronic inflammatory cell infiltration and fibrosis (Figure 1c), and became wider and severer during the experimental period (Figure 1d). Radiationinduced tubulointerstitial injuries were more severe after the sixth month of irradiation. Vascular changes consisted of intimal thickening of arteries and hyalinous thickening of arteriolar walls. The changes of vessels appeared later than those of glomeruli and tubulointerstitium. The vascular lesions were developed markedly at the end of the experimental period.Table 1 Grade of Histological Change of Each Component in Various Groups
Note:-, no lesion; ±, traced lesion; +, mild; ++, moderate; +++, severe. See test for details
ImmunohistochemistryThe patterns of immunochemical staining in different components of the kidney are summarized in Table 2. Type III collagen was weakly stained in the control rat kidneys (Figure 2a). Markedly increased expression of type III collagen was noted in widen interstitial fibrosis in radiation nephropathy (Figure 2b). Control kidneys showed mild but distinct expression of type IV collagen in glomerular basement membrane and tubular basement membrane in irradiated rat kidneys (Figure 3a). 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 3b). In the control rat kidneys, vimentin was weakly positive in the glomeruli, but negative in tubular epithelial cells (Figure 4a). In radiation nephropathy, tubular epithelial cells and interstitial cells in and around the fibrous areas showed strong immunostaining for vimentin (Figure 4b), and was parallel to the progression of tubulointerstitial fibrosis of radiation nephropathy. AlphaSMA was present mainly in the vascular wall in the control rat kidneys (Figure 5a). An increased number of interstitial cells expressed α-SMA in kidneys obtained from irradiated rats (Figure 5b). The expression of α-SMA was markedly elevated from the first, third month after radiation (1.70±0.27 vs 0.80±0.27, 2.30±0.27 vs 0.80±0.27; P<0.05, respectively). Significantly increased expression of α-SMA was noted at the sixth and ninth month after radiation (4.10±0.22 vs 0.80±0.27, 4.80±0.27 vs 0.90±0.22;P<0.001, respectively), and it was parallel to glomerulosclerosis and tubulointerstitial fibrotic process.Table 2 Grade of Immunohistochemical Staining in Kidneys 9 Months after Irradiation
Note:±, traced lesion; +, mild; ++, moderate; +++, severe. See text for details
Double ImmunostainingBy double immunostaining, coexpression of both vimentin and α-SMA was present in the tubular epithelial cells of irradiated rat kidneys. Frequently α-SMApositive myofibroblasts in the interstitium were also noted to have vimentinpositive expression in radiation nephropathy (Figure 6).
DISCUSSION
The pronounced radiosensitivity of renal tissue limits the total radiotherapeutic treatment. In our study, radiationinduced chronic renal injury consisted of 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 induced by radiation exposure was occurred timedependent. The progression of chronic renal failure stems essentially from tubulointerstitial fibrosis. How a single exposure of irradiation leads to structural changes in the kidneys is not yet clear. 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. We have previously reported that upregulation of HSP47 in radiation nephropathy may contribute to the tubulointerstitial injuries, possibly by regulating increased production of collagens [2]. Recent studies have suggested that tubular epithelial cells have the ability to transdifferentiate after injurious stimulus and are capable of producing interstitial collagens, and α-SMA might play a significant role in EMT [5, 6]. EMT is a complex process in which renal tubular cells lose their polarized tubular epithelial phenotype and acquire new features characteristic of myofibroblasts, the major effector cells responsible for the excess deposition of interstitial ECM under pathologic conditions [6, 7]. Tubular epithelial cells lose Ecadherin, an adhesive junction protein expressed in differentiated and polarized epithelial cells, and acquire the myofibroblast marker α-SMA [7]. As the major marker of EMT, α-SMA is an actin isoform that contributes to cellgenerated mechanical tension, is normally restricted to cells of vascular smooth muscle, but α-SMA can also be expressed in certain nonmuscle cells, most notably myofibroblasts. In addition to its importance as a structural protein in tissue remodeling and contraction, α-SMA may serve as a mechanotransducer, based on its ability to physically link mechanosensory elements and to enhance its own, forceinduced expression [8]. Irrespective of the initial causes, interstitial fibrosis is a remarkably monotonous process characterized by de novo activation of α-SMA positive myofibroblasts, the principal effector cells that are responsible for the excess deposition of interstitial ECM under pathologic conditions [9]. Studies of Strutz,et al. [10] indicate that tubular epithelial cells could express fibroblast markers in disease states, postulating a possibility of EMT. The features of myofibroblast include: a spindled cell morphology, an abundant matrix, immunostaining for α-SMA, rough endoplasmic reticulum, peripherally located smooth muscle type myofilaments, a Golgi apparatus producing collagensecretion granules, gap junctions and fibronexus junctions [11]. Myofibroblasts are thought to represent the main source of increased ECM deposition in renal fibrosis. It was widely considered that myofibroblasts originate from transdifferention of renal interstitial fibroblasts, pericyte of vessel and smooth muscle cells. While the role of myofibroblasts in renal fibrosis is widely accepted, their origins and activation process in the tubulointerstitial fibrosis remain largely undefined and controversial.
The main finding of our study was that α-SMA was strongly expressed in tubulointerstitial fibrosis and glomerulosclerosis in the sections of Xray irradiated rat kidneys, as detected by immunohistochemistry. Markedly expression of α-SMA from the first, third month after radiation, was noted at early stage of radiation nephropathy. Significantly increased expression of α-SMA was observed at sixth, ninth month after radiation. Moreover, it correlated closely with the increased deposition of type Ⅲ and type Ⅳ collagens, and was parallel to glomerulosclerosis and tubulointerstitial fibrotic processing in radiation nephropathy. These increases in ECM components were accompanied by a significant increase in interstitial α-SMA, suggesting activation of interstitial fibroblasts into myofibroblasts. We also observed that increased expression of vimentin in tubular epithelial cells and around interstitial fibrous areas, was parallel to the progression of tubulointerstial fibrosis of radiation nephropathy. In this study, utilizing a double immunohistochemistry technique, we were able to show colocalization of α-SMA and vimentin in tubular epithelial cells, suggested that tubular epithelial cells after radiation underwent EMT. In the interstitium, α-SMApositive myofibroblasts still coexpressed with vimentin immunostaining in radiation nephropathy, furthermore suggested that tubular epithelial cells transdifferentiated, and closely related to the process of tubulointerstitial fibrosis. We demonstrated that there were abundant cells coexpressing both α-SMA and tubular marker vimentin, indicating that they are at transitional stage between epithelia and mesenchyme. Using a rat model of radiation nephropathy, we investigated role of α-SMA at different time points and the relations with other cell phenotype, in order to make further step in exploring the relations between tubular EMT and interstitial fibrosis.
Phenotypic alteration of tubulointerstitial cells might be caused by irradiation, such as α-SMApositive interstitial cells and vimentinpositive tubular epithelial cells. Alphasmooth muscle actin is widely used to identify phenotypically altered renal interstitial cells and glomerular mesangial cells, while vimetin is used for identifying damaged renal tubular epithelial cells [2~4]. The present study revealed accumulation of collagens typeⅢ and type Ⅳ in and around the fibrotic areas. It might be contributed by phenotypically altered cells, such as vimentin positive tubular regenerative cells and α-SMA positive myofibroblasts. These findings suggest that tubular epithelial cells and interstitial fibroblasts are both active participants in the development of radiationinduced renal fibrosis. Although the mechanisms involved in the radiationinduced activation of interstitial fibroblasts and tubular epithelial cells are not well elucidated, increasing evidence supports the concept of progenitor fibroblasts to postmitotic fibroblast cell system. Thus, radiation appears to cause a shift in the ratio of progenitor fibroblasts to postmitotic fibrocytes. The latter cell type is characterized by a high capacity for synthesis of interstitial collagens which leads to the fibrotic phenotype observed in the kidney and other lateresponding tissues after irradiation [12, 13]. It has been shown that radiation exposure could initiate inflammatory events, releasing various cytokines, chemokines (such as IL-1, Il-4, MCP-1, osteopontine and RANTES) and growth factors, all of these early secreted products have been shown to link to later fibrotic events and various fibrotic renal diseases [13~15]. In this study, we showed that overexpression of α-SMA in tubulointerstitial fibrosis and glomerulosclerosis in irradiated rat kidneys, and carrying on increase along with the progression of radiation nephropathy. These findings indicate that α-SMApositive myofibroblasts may be an important media in the process of renal fibrosis of radiation nephropathy. In this sense, a key to an effective therapy for radiation nephropathy is to find a strategy that inhibits the proliferation of renal myofibroblasts in damaged kidney.
In conclusion, we explored the relations between the α-SMA expression and tubular epithelial cells phenotype in radiation nephropathy. Our results shown that increased expression of α-SMA was noted in early stage of irridiated kidneys, and the degree of α-SMA expression closely correlated with the progression of tubulointerstitial fibrosis. Tubular EMT plays a key role in the initiation and development of renal fibrosis in radiation nephropathy. Therefore, detection of α-SMA expression could be used as an indication to estimate the degree of renal fibrosis, and may be an early prognostic marker of fibrotic process in radiation nephropathy.
ACKNOWLEDGEMENT
We thank all technical staff of the Department of Nephrology, Zhong Da Hospital, Southern University School of Medicine.
REFERENCES
1. Jinde K, Nikolic Paterson DJ, Huang XR, et al. Tubular phenotypic change in progressive tubulointerstitial fibrosis in human glomerulonephritis. Am J Kidney Dis, 2001, 38(4):761.
2. Liu DG, Mohammed S, Arifa N, et al. Role of heat shock protein 47 on tubulointerstitium in experimental radiation nephropathy. Pathology International, 2002, 52:340-347.
3. Razzaque MS, Taguchi T. The possible role of colligin/HSP 47, a collagenbinding protein, in the pathogenesis of human and experimental fibrotic disease. Histol. Histopathol, 1999, 14: 1199-1212.
4. Liu DG, Razzaque MS, Cheng M, et al. The renal expression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats. The Histochemical Journal, 2001, 33:621-628.
5. Ng YY, Huang TP, Yang WC, et al. Tubular epithelialmyofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats. Kidney Int, 1998, 54:864-876.
6. Hewitson TD, Wu HL, Becker GJ. Interstitial myofibroblasts in experimental renal infection and scarring. Am J Nephrol, 1995, 15:411-417.
7. Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol, 2004, 15(1):1-12.
8. Wang J, Zohar R, McCulloch CA. Multiple roles of alphasmooth muscle actin in mechanotransduction. Exp Cell Res, 2006, 312(3):205-214.
9. Lee JM, Dedhar S, Kalluri R, et al. The epithelialmesenchymal transition: new insights in signaling, development, and disease. J Cell Biol, 2006, 172(7):973-981.
10. Strutz F, Okada H, Lo CW, et al. Identification and characterization of a fibroblast marker: FSP1. J Cell Biol, 1995, 130(2):393.
11. Eyden B. The myofibroblast: a study of normal, reactive and neoplastic tissues, with an emphasis on ultrastructure. Part 1-normal and reactive cells. J Submicrosc Cytol Pathol, 2005, 37(2):109-204.
12. Cohen EP, Robbins ME. Radiation nephropathy. Semin Nephrol, 2003, 23(5):486-499.
13. Moulder JE, Cohen EP. Radiationinduced multiorgan involvement and failure: the contribution of radiation effects on the renal system. BJR Suppl, 2005, 27:82-88.
14. Zeisberg M, Strutz F, Muller GA. Renal fibrosis: an update. Curr Opin Nephrol Hypertens, 2001, 10:315-320.
15. Robbins ME, Diz DI. Pathogenic role of the reninangiotensin system in modulating radiationinduced late effects. Int J Radiat Oncol Biol Phys, 2006, 64(1):6-12.
(Editor Emilian)(LIU Diange, ZHANG Qingj)
Department of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, Jiangsu Province 210009, China
Correspondence to LIU Diange, MD, PhD,Department of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Dingjiaqiao 87, Nanjing, Jiangsu Province 210009, China
Tel: +86-25-83285139，Email: liudiange2003＠yahoo.com. cn
[Abstract] Objective The molecular mechanisms of fibrosis in radiation nephropathy have received scant attention. However, the precise mechanism of tubulointerstitial injury in this condition is still unclear. Glomerular and tubulointerstitial α-smooth muscle actin (α-SMA) have been associated with progressive glomerulosclerosis and tubulointerstitial fibrosis. We observed expression and significance of α-SMA on the progression of radiation nephropathy in rats. Methods The model of radiation nephropathy with rats were established as follows: control group (n=12)；irradiated group (n=20), after right nephrectomy, a single dose 25 Gy Xray to left kidney. The rats were followed up for 1, 3, 6 and 9 months after renal exposure to radiation. The kidneys were examined by immunohistochemical method including double immunostaining.
Results Renal dysfunction was noted early in irradiated rats, along with inflammatory cell infiltration and interstitial fibrosis. Compared to control rat kidneys, increased expression of α-SMA and vimentin were noted in the early stage in irradiated kidneys. Further, expression of α-SMA was carrying on increase along with the progression of radiation nephropathy. Double immunostaining demonstrated tubular epithelial cell with vimentinpositive expression coexisted expression of α-SMA. Moreover, α-SMApositive myofibroblasts in the interstitium also appeared vimentinpositive expression. These findings showed that there were abundant cells coexpressing both α-SMA and tubular marker, indicating that they are at transitional stage between epithelial and mesenchyme. Overexpression of α-SMA and vimentin were closely associated with increased deposition of type Ⅲ and Ⅳ collagens in the widened interstitium of irradiated rats. Conclusions The phenotype change tubular epithelial cell possibly induced renal resident cell to occur the transdifferentiation, and participated in the development of fibrotic event in radiation nephropathy. The α-SMA might be regarded as index of early prognosis and determination of tubulointerstitial fibrosis in radiation nephropathy.
[Key words] α-smooth muscle actin; vimentin; mmunohistochemistry; radiation nephropathy;tubulointerstitial fibrosis
INTRODUCTION
Sufficient irradiation on the kidney causes progressive injury results in fibrotic process and organ failure. This injury is an example of normal tissue radiation injury, which is a doselimiting problem for all therapeutic irradiation. More studies have focused on a primary role for glomerular cells and tubular epithelial cells in radiationinduced renal injury. Especially in chronic phase of radiation nephropathy, renal fibrosis resulted from increased accumulation of various extracellular matrices (ECM) is a prominent feature. However, the particular cell types, mediators and mechanisms involved are still ill defined. Activation α-SMApositive myofibroblasts is considered a key event in the progression of renal fibrosis. Under pathologic conditions, tubular epithelial cells may transdifferentiate into myofibroblastsby a conversion known as tubular epithelialmesenchymal transition (EMT) [1], and participate in the development of tubulointerstitial fibrosis. We have reported that role of heat shock protein 47 (HSP47) in experimental radiation nephropathy, upregulation of HSP47 in phenotypically altered renal cells might contribute to synthesis of collagens resulting in tubulointerstitial fibrosis in radiation nephropathy [2]. The present studies used a rat model of radiation nephropathy to investigate expression and significance of α-SMA on the progression of radiation nephropathy. This study was focused on colocalization of vimentin and α-SMA by double immunostaining method, in order to elucidate the role of α-SMA on the progression of radiation nephropathy.
The results suggest that phenotypically altered renal cells might contribute to synthesis of collagens resulting in tubulointerstitial fibrosis in radiation nephropathy. The α-SMA might be regarded as an early marker of determination of tubulointerstitial fibrosis in radiation nephropathy.
METHODS
Experimental AnimalsMale Wistar rats (n=32), aged 6 weeks, were divided into two experimental groups. Radiation procedures were previously reported [2]. Briefly, control group (n=12) included agematched control rats received only laparotomy without irradiation. For rats in irradiated group (n=20), after intraperitoneal nembutal anesthesia (25 mg/kg of body weight), were nephrectomied for their right kidneys before radiation procedures. The left kidney was exposed through the surgical incision and covered with sterile gauze saturated with physiologic saline solution. The exposed left kidney was a singledose 25 Gy Xray irradiated through a specially designed lead shield, which protected the entire body and only allowed irradiation of left kidney. The irradiation source was provided by a 200 kV, 15 mV, superficial therapy unit (filter 0.05Al, Xray machine, Toshiba, Tokyo, Japan)，through the distance from target to organ measuring 50 cm. After treatment, the kidney was restored to its original position, and animals were given a regular diet and water. All the experimental procedures followed the guideline for care and use of laboratory animals.
Renal Function StudiesBlood was collected from the superior vena cava from different group of rats, at various time points, when the rats were killed. The levels of blood urea nitrogen (BUN) and serum creatinine (Cr) were measured by autoanalyzer (Hitachi 7170, Hitachi City, Japan).
Tissue CollectionAt each day of the first, third, sixth and ninth month after radiation, three rats of control group and five rats of irradiated group were examined respectively. The kidneys were rapidly removed and weighed, the fixed immediately in 10% formalin for 24 h and processed further for histological and immunohistochemical examination.
Histological StudiesTissue were processed and embedded in paraffin, cut into 4 μm sections and stained with hematoxylineosin (HE), periodic acidSchiff (PAS), periodic acidmethenamine sliver (PAM) and Masson trichrome. The extent of glomerular, tubulointerstitial and vascular damages was examined by light microscopy[2]. Glomeruli were scored individually based on the averaged percentage of sclerotic area of glomerular tuft as follows: -, no sclerosis; ±, sclerotic area <5%; +, sclerotic area 5%~25%; ++, sclerotic area 25%~50%; +++, sclerotic area >50%. Tubulointerstitial injuries were semiquantified using PASstained sections and were scored as follows: -, no injury; ±, injury involved <5%; +, injury involved 5%~25%; ++, injury involved 25%~50%; +++, injury involved >50%. The vascular wall thickening and luminal narrowing as follows: -, no thickening; +, mild thickening; ++, moderate thickening; +++, severe thickening.
ImmunohistochemistryImmunohistochemical methods were performed as described elsewhere [2~4]. Briefly, paraffin sections (4 μm) were deparaffinized with xylene, rinsed thoroughly with ethanol, then soaked in 0.3% hydrogen peroxide in methanol for 30 min at room temperature to inactivate endogenous peroxidase activity. After mild treatment with 0.05% trypsin (T 4799, Sigma, St Luis, MO, USA) for 5 min, the sections were incubated with either 10% goat serum or 10% rabbit serum for 30 min, then covered with primary antibodies, washed with phosphatebuffered saline (PBS) and processed further using Histofine SABPO 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: α-SMA (Dako, Glostrup, Denmark), vimentin (Dako), type Ⅲ collagen (Chemicon, Temecula, CA, USA), type Ⅳ collagen (Chemicon). The staining intensity was graded semiquantitatively according to the following scales [2]: -, no staining; ±, staining area <5%; +, staining area 5%~25%; ++, staining area 25%~50%; +++, staining area >50%. The extent of α-SMA immunostaining was scored as previously reported [1]: 0, negative; 1, traced (<5%)；2, mild (5%~25%)；3, moderate (25%~50%)；4, severe (>50%). For each group of slides, five adjacent eyepiece grid areas were studied, starting just below the renal capsule, and proceeding toward the medulla. A 100point eyepiece grid was used for point counting (Zeiss, Welsyn Garden City, UK). Scores were calculated for at least 30 eyefields at magnification 400, summed, and averaged for each time point.
Double ImmunostainingDouble immunostaining for vimentin and α-SMA was performed as described earlier [2~4]. Sections were processed as above except that the sections were incubated first with the monoclonal antibody against vimentin for 1 h, then with biotinylated second antibody for 10 min and streptavidinalkaline phosphatase for 10 min, and developed with 5bromo-4chloro3indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT), which produced a dark purple stain. Then the vimentinstained slides were counterstained with α-SMA by the streptavidinbiotinperoxidase method and antigenantibody complex was visualized by aminoethyl carbazole (AEC)/ H2O2, which produced an intense red stain. In between the first and second staining, the sections were autoclaved in citrate buffer (pH 6.0) for 15 min to minimize the cross reactivity between the first and second staining. As immunohistochemical control, primary antibodies were replaced with either 0.01 M PBS or mouse IgG/rabbit IgG diluted with PBS in a concentration similar to that of the primary antibodies.
Statistical Analysis
Statistical analysis was carried out using StatViewJ5.0. The data are shown as a mean ± SD. Comparisons between time points were made by first testing for group differences by Students ttest. Statistical significance was achieved at corrected P<005.
RESULTS
Blood ChemistryThe level of BUN and Cr in irradiated group began to markedly increase from the first, third month after irradiation (BUN: 27.7±0.31 vs 16.5±0.20 mg/dl, 44.3±0.38 vs 16.8±0.32; Cr: 0.49±0.08 vs 021±0.07 mg/dl, 0.62±0.08 vs 0.23±0.06 mg/dl; P<0.05, respectively). Compared to the control group, the level of BUN and Cr were significantly raised at the sixth and ninth month after irradiation (BUN: 98.5±0.40 vs 17.9±0.26 mg/dl, 192.3±4.78 vs 18.5±0.20 mg/dl; Cr: 1.42±0.07 vs 0.26±0.09 mg/dl, 2.68±0.08 vs 0.31±0.08 mg/dl; P<0.001, respectively). The renal function was parallel to the histological behaviors.
MorphologyTable 1 details the light microscopic morphological changes of kidneys at different times in 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 lesion at early stage (Figure 1b). The glomerular lesion gradually developed global glomerulosclerosis in time course (Figure 1c, d). The damages of tubular epithelium were composed of cellular degeneration, atrophy, dilatation and thickening of tubular basement membrane, tubular hyalinous casts were also noted in places in irradiated rat kidneys, the interstitium showed chronic inflammatory cell infiltration and fibrosis (Figure 1c), and became wider and severer during the experimental period (Figure 1d). Radiationinduced tubulointerstitial injuries were more severe after the sixth month of irradiation. Vascular changes consisted of intimal thickening of arteries and hyalinous thickening of arteriolar walls. The changes of vessels appeared later than those of glomeruli and tubulointerstitium. The vascular lesions were developed markedly at the end of the experimental period.Table 1 Grade of Histological Change of Each Component in Various Groups
Note:-, no lesion; ±, traced lesion; +, mild; ++, moderate; +++, severe. See test for details
ImmunohistochemistryThe patterns of immunochemical staining in different components of the kidney are summarized in Table 2. Type III collagen was weakly stained in the control rat kidneys (Figure 2a). Markedly increased expression of type III collagen was noted in widen interstitial fibrosis in radiation nephropathy (Figure 2b). Control kidneys showed mild but distinct expression of type IV collagen in glomerular basement membrane and tubular basement membrane in irradiated rat kidneys (Figure 3a). 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 3b). In the control rat kidneys, vimentin was weakly positive in the glomeruli, but negative in tubular epithelial cells (Figure 4a). In radiation nephropathy, tubular epithelial cells and interstitial cells in and around the fibrous areas showed strong immunostaining for vimentin (Figure 4b), and was parallel to the progression of tubulointerstitial fibrosis of radiation nephropathy. AlphaSMA was present mainly in the vascular wall in the control rat kidneys (Figure 5a). An increased number of interstitial cells expressed α-SMA in kidneys obtained from irradiated rats (Figure 5b). The expression of α-SMA was markedly elevated from the first, third month after radiation (1.70±0.27 vs 0.80±0.27, 2.30±0.27 vs 0.80±0.27; P<0.05, respectively). Significantly increased expression of α-SMA was noted at the sixth and ninth month after radiation (4.10±0.22 vs 0.80±0.27, 4.80±0.27 vs 0.90±0.22;P<0.001, respectively), and it was parallel to glomerulosclerosis and tubulointerstitial fibrotic process.Table 2 Grade of Immunohistochemical Staining in Kidneys 9 Months after Irradiation
Note:±, traced lesion; +, mild; ++, moderate; +++, severe. See text for details
Double ImmunostainingBy double immunostaining, coexpression of both vimentin and α-SMA was present in the tubular epithelial cells of irradiated rat kidneys. Frequently α-SMApositive myofibroblasts in the interstitium were also noted to have vimentinpositive expression in radiation nephropathy (Figure 6).
DISCUSSION
The pronounced radiosensitivity of renal tissue limits the total radiotherapeutic treatment. In our study, radiationinduced chronic renal injury consisted of 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 induced by radiation exposure was occurred timedependent. The progression of chronic renal failure stems essentially from tubulointerstitial fibrosis. How a single exposure of irradiation leads to structural changes in the kidneys is not yet clear. 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. We have previously reported that upregulation of HSP47 in radiation nephropathy may contribute to the tubulointerstitial injuries, possibly by regulating increased production of collagens [2]. Recent studies have suggested that tubular epithelial cells have the ability to transdifferentiate after injurious stimulus and are capable of producing interstitial collagens, and α-SMA might play a significant role in EMT [5, 6]. EMT is a complex process in which renal tubular cells lose their polarized tubular epithelial phenotype and acquire new features characteristic of myofibroblasts, the major effector cells responsible for the excess deposition of interstitial ECM under pathologic conditions [6, 7]. Tubular epithelial cells lose Ecadherin, an adhesive junction protein expressed in differentiated and polarized epithelial cells, and acquire the myofibroblast marker α-SMA [7]. As the major marker of EMT, α-SMA is an actin isoform that contributes to cellgenerated mechanical tension, is normally restricted to cells of vascular smooth muscle, but α-SMA can also be expressed in certain nonmuscle cells, most notably myofibroblasts. In addition to its importance as a structural protein in tissue remodeling and contraction, α-SMA may serve as a mechanotransducer, based on its ability to physically link mechanosensory elements and to enhance its own, forceinduced expression [8]. Irrespective of the initial causes, interstitial fibrosis is a remarkably monotonous process characterized by de novo activation of α-SMA positive myofibroblasts, the principal effector cells that are responsible for the excess deposition of interstitial ECM under pathologic conditions [9]. Studies of Strutz,et al. [10] indicate that tubular epithelial cells could express fibroblast markers in disease states, postulating a possibility of EMT. The features of myofibroblast include: a spindled cell morphology, an abundant matrix, immunostaining for α-SMA, rough endoplasmic reticulum, peripherally located smooth muscle type myofilaments, a Golgi apparatus producing collagensecretion granules, gap junctions and fibronexus junctions [11]. Myofibroblasts are thought to represent the main source of increased ECM deposition in renal fibrosis. It was widely considered that myofibroblasts originate from transdifferention of renal interstitial fibroblasts, pericyte of vessel and smooth muscle cells. While the role of myofibroblasts in renal fibrosis is widely accepted, their origins and activation process in the tubulointerstitial fibrosis remain largely undefined and controversial.
The main finding of our study was that α-SMA was strongly expressed in tubulointerstitial fibrosis and glomerulosclerosis in the sections of Xray irradiated rat kidneys, as detected by immunohistochemistry. Markedly expression of α-SMA from the first, third month after radiation, was noted at early stage of radiation nephropathy. Significantly increased expression of α-SMA was observed at sixth, ninth month after radiation. Moreover, it correlated closely with the increased deposition of type Ⅲ and type Ⅳ collagens, and was parallel to glomerulosclerosis and tubulointerstitial fibrotic processing in radiation nephropathy. These increases in ECM components were accompanied by a significant increase in interstitial α-SMA, suggesting activation of interstitial fibroblasts into myofibroblasts. We also observed that increased expression of vimentin in tubular epithelial cells and around interstitial fibrous areas, was parallel to the progression of tubulointerstial fibrosis of radiation nephropathy. In this study, utilizing a double immunohistochemistry technique, we were able to show colocalization of α-SMA and vimentin in tubular epithelial cells, suggested that tubular epithelial cells after radiation underwent EMT. In the interstitium, α-SMApositive myofibroblasts still coexpressed with vimentin immunostaining in radiation nephropathy, furthermore suggested that tubular epithelial cells transdifferentiated, and closely related to the process of tubulointerstitial fibrosis. We demonstrated that there were abundant cells coexpressing both α-SMA and tubular marker vimentin, indicating that they are at transitional stage between epithelia and mesenchyme. Using a rat model of radiation nephropathy, we investigated role of α-SMA at different time points and the relations with other cell phenotype, in order to make further step in exploring the relations between tubular EMT and interstitial fibrosis.
Phenotypic alteration of tubulointerstitial cells might be caused by irradiation, such as α-SMApositive interstitial cells and vimentinpositive tubular epithelial cells. Alphasmooth muscle actin is widely used to identify phenotypically altered renal interstitial cells and glomerular mesangial cells, while vimetin is used for identifying damaged renal tubular epithelial cells [2~4]. The present study revealed accumulation of collagens typeⅢ and type Ⅳ in and around the fibrotic areas. It might be contributed by phenotypically altered cells, such as vimentin positive tubular regenerative cells and α-SMA positive myofibroblasts. These findings suggest that tubular epithelial cells and interstitial fibroblasts are both active participants in the development of radiationinduced renal fibrosis. Although the mechanisms involved in the radiationinduced activation of interstitial fibroblasts and tubular epithelial cells are not well elucidated, increasing evidence supports the concept of progenitor fibroblasts to postmitotic fibroblast cell system. Thus, radiation appears to cause a shift in the ratio of progenitor fibroblasts to postmitotic fibrocytes. The latter cell type is characterized by a high capacity for synthesis of interstitial collagens which leads to the fibrotic phenotype observed in the kidney and other lateresponding tissues after irradiation [12, 13]. It has been shown that radiation exposure could initiate inflammatory events, releasing various cytokines, chemokines (such as IL-1, Il-4, MCP-1, osteopontine and RANTES) and growth factors, all of these early secreted products have been shown to link to later fibrotic events and various fibrotic renal diseases [13~15]. In this study, we showed that overexpression of α-SMA in tubulointerstitial fibrosis and glomerulosclerosis in irradiated rat kidneys, and carrying on increase along with the progression of radiation nephropathy. These findings indicate that α-SMApositive myofibroblasts may be an important media in the process of renal fibrosis of radiation nephropathy. In this sense, a key to an effective therapy for radiation nephropathy is to find a strategy that inhibits the proliferation of renal myofibroblasts in damaged kidney.
In conclusion, we explored the relations between the α-SMA expression and tubular epithelial cells phenotype in radiation nephropathy. Our results shown that increased expression of α-SMA was noted in early stage of irridiated kidneys, and the degree of α-SMA expression closely correlated with the progression of tubulointerstitial fibrosis. Tubular EMT plays a key role in the initiation and development of renal fibrosis in radiation nephropathy. Therefore, detection of α-SMA expression could be used as an indication to estimate the degree of renal fibrosis, and may be an early prognostic marker of fibrotic process in radiation nephropathy.
ACKNOWLEDGEMENT
We thank all technical staff of the Department of Nephrology, Zhong Da Hospital, Southern University School of Medicine.
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(Editor Emilian)(LIU Diange, ZHANG Qingj)