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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 59:B984-B996 (2004)
© 2004 The Gerontological Society of America

Expression and Significance of Integrin-Linked Kinase in Cultured Cells, Normal Tissue, and Diseased Tissue of Aging Rat Kidneys

Zhihui Li1, Xiangmei Chen1,, Yuansheng Xie1, Suozhu Shi1, Zhe Feng1, Bo Fu1, Xiaojie Zhang1, Guangyan Cai1, Chuanyue Wu2, Di Wu1 and Yunjuan Gu1

1 Department of Nephrology, Kidney Center & Key Lab of PLA, Chinese General Hospital of PLA, Beijing.
2 Department of Pathology, University of Pittsburgh, Pennsylvania.

Address correspondence to Xiangmei Chen, MD, PhD, Department of Nephrology, Kidney Center and Key Lab of PLA, Chinese General Hospital of PLA, Fuxing Road 28, Beijing 100853, P.R. China. E-mail: xmchen{at}public.bta.net.cn


   Abstract
Top
 Abstract
Experimental Procedures
 Results
 Discussion
 References
 
Integrin-linked kinase (ILK) is an integrin-binding cytoplasmic protein that has been implicated in regulating numerous cellular processes and fibronectin (Fn) deposition through mediated integrin, but the expression and significance of ILK in the aging kidney have not yet been reported. We report here that mRNA and protein expression of ILK increased in primary cultured mesangial and tubular epithelial cells, and normal and unilateral ureteral obstructed kidney tissues in 28-month-old rats but not in 3-month-old rats, moreover, accompanied by the over-expression of Fn and integrin-ß1 in the aging kidney, by means of Northern blot, Western blot, and immunofluorescent double-staining immunohistochemistry. In addition, in the primary cultured kidney cells, ILK expression was positively correlated with senescence-associated ß-gal positive staining and negatively correlated with cellular proliferation. The results suggest that ILK may be involved in the fibrotic or senescent process in the aging kidney.

AGING is a complex physiological process in which the function and structure of many organ systems become altered. Glomerulosclerosis and tubulointerstitial fibrosis are frequently present in kidneys from aged animals and humans (1). Many studies have suggested that glomerulosclerosis and tubulointerstitial fibrosis in the aging kidney were closely correlated with fibronectin (Fn) deposited into extracellular matrix (1–4). But why is extracellular matrix abnormally deposited in the aging kidney? The mechanisms are unclear.

Integrin-linked kinase (ILK) is an integrin-binding cytoplasmic protein that interacts with the cytoplasmic domains of ß-integrins and numerous cytoskeleton-associated proteins (5–8). ILK has been shown to be involved in the regulation of a number of integrin-mediated processes and extracellular matrix accumulation (9–14). Recent studies have shown that over-expression of ILK in cultured epithelial cells dramatically stimulates the deposition of fibronectin into the extracellular matrix, because the over-expression of a kinase-inactive ILK mutant failed to enhance the matrix assembly, identifying ILK as an important regulator of pericellular Fn matrix assembly (15). Guo and Li have demonstrated that ILK expression increases in human diabetic nephropathy and in the unilateral ureteral obstruction (UUO) model mouse, which results in increased deposition of Fn in kidneys (16,17). But up to now, the expression and significance of ILK in aging kidney have not been reported. Whether or not the expression of ILK, integrin, and Fn and their relationship would change in the aging kidney has not been studied.

In this study, we investigated the expression of ILK in the kidneys of normal and UUO rats, as well as cultured mesangial and tubular epithelial cells in 3-month-old and 28-month-old rats using Northern blot, Western blot, and immunofluorescent double-staining immunohistochemistry. Our results suggest that ILK might be closely correlated with the fibrotic process or senescent process in the aging kidney.


   EXPERIMENTAL PROCEDURES
Top
 Abstract
 Experimental Procedures
Results
 Discussion
 References
 
Antibodies and Reagents
Mouse anti-ILK monoclonal antibody (Ab) 65.1 was generated as previously described (18) and used for immunostaining. The rabbit anti-ILK polyclonal Ab was purchased from Upstate Biotechnology (Charlottesville, VA) and used for Western blot. The goat antiintegrin-ß1 polyclonal Ab and rabbit antifibronectin polyclonal Ab were purchased from BD Pharmingen (San Jose, CA). Secondary Abs was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cell culture media, fetal bovine serum (FBS), and supplements were obtained from the Invitrogen Corporation (Carlsbad, CA). All other chemicals were of analytic grade and were obtained from Sigma-Aldrich (St. Louis, MO).

Cell Culture and Treatment
Rat glomerular mesangial cells and tubular epithelial cells were cultured as described previously (19,20). Briefly, the kidneys of eight 3-month-old male Wistar rats weighing 200–250 g and eight 28-month-old rats weighing 470–530 g were removed and decapsulated under sterile conditions. Cortical tissue was cut away from the medulla and minced in isolation buffer solution. Glomeruli and tubules were isolated by sequential sieving and collected. After incubation with 0.1% collagenase in isolation buffer solution for 30 minutes at 37°C, glomeruli and tubules were centrifuged and collected. Isolated glomeruli were plated in RPMI 1640 medium supplemented with 17% FBS, 100 units/ml of penicillin, 100 units/ml of streptomycin, 5 µg/ml of insulin, 5 µg/ml of transferrin, and 5 ng/ml of selenite. Isolated tubular epithelial cells were cultured in DMEM medium, and supplemented with reagents as above. Desmins and cytokeratins were monitored as glomerular mesangial cells and tubular epithelial cell markers by means of indirect immunofluorescence staining, respectively. Glomerular mesangial cells and tubular epithelial cells were collected and used for senescence-associated (SA)-ß-gal staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, indirect immunofluorescence staining, and Northern blot analysis.

Animal Model
To study ILK expression in normal young and aging kidneys, eight 3-month-old male Wistar rats weighing 200–250 g and eight 28-month-old rats weighing 470–530 g were killed, and their kidneys were removed. One part of each kidney was fixed in 10% phosphate-buffered saline (PBS) formalin, followed by paraffin embedding for histologic and immunohistochemical studies. Another part was immediately frozen in Tissue-Tek OCT compound (Sakura Finetek, Inc., Torrance, CA) for cryosection. The remaining kidneys were snap-frozen in liquid nitrogen and stored at –80°C for protein and RNA extractions.

To study ILK expression in aging kidneys with renal interstitial fibrosis, a UUO rat model was performed using an established procedure as described elsewhere (21,22). The thirty-two 3-month-old male Wistar rats weighing 200–250 g and the thirty-two 28-month-old rats weighing 470–530 g were used in the present study. Both 3- and 28-month old male Wistar rats were randomly divided into a control group (sham-operated rats, 8 young rats, and 8 aging rats) and a UUO group (24 young rats and 24 aging rats). In rats of the UUO group, under general anesthesia with pentobarbital sodium (35 mg/kg body wt), complete ureteral obstruction was carried out by double-ligating the left ureter using 4-0 silk after a midline abdominal incision. Sham-operated rats had their ureters exposed and manipulated but not ligated. Then, the animals of UUO group were randomly subdivided into days 3, 7, and 14 groups. Eight rats from each group were killed at different time point, and their kidneys were removed. Kidney morphology and renal ILK expression were analyzed.

Senescence-Associated-ß-Gal Staining in Mesangial and Tubular Epithelial Cells
Glomerular mesangial cells and tubular epithelial cells of 3- and 28-month-old rats, respectively, were cultured in complete medium on plates to reach 70% confluence. Cells were washed twice with PBS, fixed with 0.2% glutaral and 2% formaldehyde in PBS for 10 minutes, then washed twice with PBS. Cells were stained with 5-bromium-4-chlorine-3-indole-ß-D-galactose (ß-gal) at a concentration of 1 mg/ml for 24 hours at room temperature. After being washed with PBS, percentages of SA-ß-gal staining positive cells, which had blue color sedimentation in their cytoplasm under the microscope, were counted.

MTT Assay
Glomerular mesangial cells were seeded into 96 well plates (2 x103 cells per well); after incubation for 24 hours, 48 hours, and 72 hours, respectively, MTT (2 mg/ml, 20 µl) was added into each well and incubated for 4 hours. Dimethyl sulphoxide (100 µl) was subsequently added to each well to dissolve the formazan crystals and the absorption at 570 nm was measured.

Indirect Immunofluorescence Staining in Cells and Tissues of Kidneys
Indirect immunofluorescence staining in cells was performed using a routine procedure. Briefly, cells cultured on cover slips were washed twice with cold PBS and fixed with 3.7% paraformaldehyde for 10 minutes at room temperature. Following three extensive washings with PBS containing 0.1% Triton X-100, the cells were blocked with 10% normal goat serum in PBS buffer for 20 minutes at room temperature, then incubated with the specific primary Abs for 60 minutes at 37°C, and incubated with species-specific fluorescein isothiocyanate-conjugated secondary Abs for 30 minutes at 37°C.

Kidney cryosections were prepared at 5 µm thickness and fixed for 10 minutes with cold methanol/acetone (1:1). After being blocked with 10% normal goat serum in PBS for 20 minutes, the sections were incubated with anti-ILK, anti-integrin-ß1, or anti-Fn Abs, respectively. As a negative control, the primary Ab was replaced with normal IgG (immunoglobulin G). In double-staining experiments, kidney cryosections were incubated with primary Abs from different species as specified in each experiment. After rinsing, the bound primary Abs were detected with species-specific fluorescein rhodamine-conjugated and fluorescein isothiocyanate-conjugated secondary Abs.

Stained cell monolayers were observed using a Bio-Rad Radiance 2000 laser confocal microscope (Bio-Rad Corp., Hercules, CA) equipped with fluorescein rhodamine and fluorescein isothiocyanate filters. Fluorescence intensity of cells and tissue sections were measured using the Laser Pix 4.0 software system (Bio-Rad). Twenty different microscopic fields of cells from each cell culture sample, 20 fields of glomeruli and 20 fields of tubulointerstitium from each kidney tissue cortex (magnification shown in respective figure) were randomly chosen for fluorescence intensity analysis. The average fluorescence intensity for each sample of cells or kidney tissue was calculated.

Immunohistochemical Staining in UUO Model Rat Kidney Tissue
Sections (3 µm) of formalin-fixed paraffin-embedded kidney tissues were treated with 0.3% hydrogen peroxide for 10 minutes at room temperature and then with 10% normal goat serum in PBS for 20 minutes at room temperature. The sections were then incubated with primary Abs overnight at 4°C, with secondary Abs for 60 minutes at room temperature, and finally with streptavidin-HRP conjugate solution for 30 minutes at room temperature. Sections were stained with a diaminobenzidine (DAB)/hydrogen peroxide substrate solution, then counterstained with hematoxylin. Meanwhile, in negative controlled experiments, the primary Abs were replaced with nonimmune normal rabbit serum. All sections were evaluated blindly by a qualified observer using a computer image analysis system. The system is composed of an Optronic Engineering DEI-750D digital output camera (Optronics, Goleta, CA) and Scion Imaging System for Windows (Scion Corporation, Frederick, MD). Twenty tubulointerstitium microscopic fields (magnification shown in respective figure) were randomly chosen from each kidney tissue cortex for quantitative analysis. After the background was erased, the positive area and the percentages of positive staining area were measured in square pixels.

Western Blot Analysis
Kidney tissue homogenates were prepared. Briefly, 50 mg of kidney tissues were homogenized in 2 ml of ice-cold RIPA extraction buffer. Kidney tissue protein was extracted. Samples were heated at 100°C for approximately 5–10 minutes before loading and separated on 10% or 6% SDS-polyacrylamide gels. After the proteins were electrotransferred to a nitrocellulose membrane, nonspecific binding to the membrane was blocked for 1 hour at room temperature with 5% nonfat milk in TBS buffer. The membranes were then incubated for 16 hours at 4°C with various primary Abs in a blocking buffer containing 5% nonfat milk. Following extensive washing in TBS buffer, the membranes were incubated with HRP-conjugated secondary Ab for 1 hour at room temperature in 5% nonfat milk dissolved in TBS. Membranes were then washed with TBS buffer, and the signals were visualized using the ECL system (Santa Cruz).

Northern Blot Analysis
Total RNA was isolated from various cells and kidney tissues using TRIZOL reagent (GIBCO BRL, Carlsbad, CA). Northern blot analysis for mRNA expression was also carried out using routine procedures. Briefly, samples of 20 µg total RNA were electrophoresed on 1.0% formaldehyde-agarose gels and then transferred to a nylon membrane by capillary blotting. Membranes were prehybridized and hybridized at 65°C for 4 hours and 16 hours, respectively. DNA probes were done using purificated polymerase chain reaction (PCR) product, then labeled 32P using a random primer labeling kit (Stratagene, La Jolla, CA) using [-32P] dCTP.

Statistical Analysis
All data analyses were performed using SPSS software (SPSS, Inc., Chicago, IL). Parametric data are reported as mean ± standard deviation (SD). Student's t test was used for statistics analysis between the two groups. Comparison among groups was conducted using analysis of variance (ANOVA). The relationship between the two variances was tested using Pearson's correlation analysis. Probability values <.05 were considered significant.


   RESULTS
Top
 Abstract
 Experimental Procedures
 Results
Discussion
 References
 
Difference of SA-ß-Gal Staining Between Young and Aging Kidney Cells
The SA-ß-gal was a hallmark of cell replicative senescence or physiologic age that is generally accepted in the world (23). To identify difference between young and aging rat kidney cells, beta-galactosidase activity of glomerular mesangial cells and tubular epithelial cells in the 3- and 28-month-old rats were detected using SA-ß-gal staining (Figure 1). Percentages of SA-ß-gal staining positive cells were 9.0% ± 2.83% versus 79.4% ± 8.89% (p <.01) in young and aging glomerular mesangial cells and 8.52% ± 2.3% versus 72.3% ± 9.21% (p <.01) in young and aging tubular epithelial cells, respectively.



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  		Figure 1. SA-ß-gal staining of glomerular mesangial cells in the 3-month-old and 28-month-old rat. The SA-ß-gal staining positive cells show blue color sedimentation in cytoplasm (A). The percentages of SA-ß-gal staining positive glomerular mesangial cells in aging rats are higher than that in young rats (B)

 
Difference of Cell Proliferation Between Young and Aging Kidney Cells
Proliferation of cells is also a good approach to demonstrate senescence besides SA-ß-gal staining. We investigated proliferation of mesangial cells between young and aging rat kidneys using an MTT assay. The value of MTT was 0.74 ± 0.12 versus 0.59 ± 0.11 (p <.01), 0.84 ± 0.10 versus 0.64 ± 0.19 (p <.01), and 0.92 ± 0.15 versus 0.62 ± 0.19 (p <.01) in young and aging glomerular mesangial cells, after being cultured for 24 hours, 48 hours, and 72 hours, respectively.

Difference of ILK Expression Between Young and Aging Kidney Cells
Previous studies have shown that in cells cultured in plastic dishes, ILK was clustered in focal adhesions (24), regions with close contacts between the substrate and the plasma membrane on the basal surface of the cells (10,11). Our study demonstrated that ILK was presented in cytoplasm (Figure 2A). Fluorescence intensity of ILK staining was 8.23 ± 3.83 and 21.38 ± 6.56 (p <.01) in young and aging mesangial cells; 3.75 ± 1.09 and 18.07 ± 3.06 (p <.01) in young and aging tubular epithelial cells, respectively. No specific staining was detected when the anti-ILK monoclonal antibody was replaced with an irrelevant mouse IgG. Consistent with the results of immunofluorescent staining, Northern blot analysis revealed that ILK mRNA expression was also increased in aging kidney cells (Figure 2B).



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  		Figure 2. Expression of integrin-linked kinase (ILK) in young and aging kidney cells. Fluorescence intensity of ILK staining in glomerular mesangial cells and tubular epithelial cells in aging rats is higher than that in young rats (A). Lanes 1 and 2 show the results of a ILK Northern blot in 3-month-old and 28-month-old glomerular mesangial cells, and lanes 3 and 4 show the results of a ILK Northern blot in 3-month-old and 28-month-old tubular epithelial cells, respectively (B). The results show that expression of ILK mRNA is significantly increased in the glomerular mesangial cells and tubular epithelial cells of aging rats (C)

 
Furthermore, Pearson's correlation analysis showed that the percentage of SA-ß-gal staining positive cells was positively correlated with the intensity of ILK fluorescence staining (r = 0.89, P <.01), the value of the MTT assay was negatively correlated with the intensity of ILK fluorescence staining (r = –0.84, p <.01) in glomerular mesangial cells.

We also detected changes of integrin-ß1 expression in glomerular mesangial cells and epithelial cells of aging rats. The results demonstrated that integrin-ß1 expression was coincident with ILK expression in kidney cells of the 3- and 28-month-old rats, and integrin-ß1 was localized on cell membranes (data not shown).

Difference of FN Expression Between Young and Aging Kidney Cells
To determine whether ILK plays a role in the regulation of Fn assembly, we analyzed the ability of assembling Fn in glomerular mesangial cells and tubular epithelial cells of young and aging rats (Figure 3A). Immunofluorescent staining revealed that glomerular mesangial cells and tubular epithelial cells of the 3-month-old rats assembled a small amount of Fn, while those of the 28-month-old rats assembled an extensive amount of Fn. Fluorescence intensity of Fn was 10.27 ± 2.87 versus 26.51 ± 5.79 (p <.01), and 10.46 ± 5.91 versus 24.31 ± 5.86 (p <.01), in young and aging glomerular mesangial cells and tubular epithelial cells, respectively. Northern blot analysis also revealed Fn mRNA expressions of aging glomerular mesangial cells and tubular epithelial cells were higher than that of young cells (Figure 3B).



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  		Figure 3. Expression of fibronectin (Fn) in young and aging kidney cells. Glomerular mesangial cells and tubular epithelial cells of the 3-month-old rats assemble a small amount of Fn, while glomerular mesangial cells and tubular epithelial cells of the 28-month-old rats assemble an extensive Fn (A). Fluorescence intensity of Fn staining in glomerular mesangial cells and tubular epithelial cells in aging rats is higher than that in young rats. Lanes 1 and 2 shows the results of Fn Northern blot in the 3-month-old and 28-month-old glomerular mesangial cells, and lanes 3 and 4 shows the results of a Fn Northern blot in the 3-month-old and 28-month-old tubular epithelial cells, respectively (B). The results show that expression of Fn mRNA is significantly increased in the glomerular mesangial cells and tubular epithelial cells of aging rats (C)

 
Difference of ILK Expression Between Young and Aging Normal Kidney Tissues
To investigate whether ILK expression is altered in the kidney tissues of aging rats, we carried out immunofluorescent staining with the monoclonal anti-ILK Ab in frozen sections of the kidney tissues from the 3- and 28-month-old rats. The results revealed that ILK was present in the glomeruli, tubules, and tubulointerstitium (Figure 4A). We compared the ILK staining of glomeruli and tubulointerstitium of the 28-month-old rats with that of the 3-month-old rats and found that ILK fluorescence intensity was significantly increased with aging. It was 5.72 ± 2.62 versus 15.79 ± 4.22 (p <.01) and 7.60 ± 2.21 versus 16.11 ± 4.41 (p <.01) in young and aging glomeruli and tubulointerstitium, respectively.



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  		Figure 4. Expression of integrin-linked kinase (ILK) in young and aging normal kidney tissues. The fluorescence intensity of ILK staining is significantly more increased in the 28-month-old glomeruli and tubulointerstitium than in the 3-month-old ones (A). (B) reveals the results of Western blotting and (C) reveals the results of Northern blotting. Lane 1 indicates the 3-month-old rat kidney; lane 2 indicates the 28-month-old rat kidney. The results of semiquantitative analysis show that the protein and mRNA expression of ILK is significantly more increased in 28-month-old kidney tissues than in young ones

 
Western blotting analyzed ILK protein expression of the 3- and 28-month-old rat kidneys (Figure 4B). The results showed that ILK abundances of aging rat kidneys were higher than that of young rat kidneys in total protein; semiquantitative values were 0.50 ± 0.06 and 0.61 ± 0.07 (p <.05) in young and aging rat kidneys, respectively.

Northern blot analysis demonstrated that the ILK mRNA expression was also increased with aging (Figure 4C). The semiquantitative value was 0.47 ± 0.07 versus 0.58 ± 0.05 (p <.05) in young and aging kidney tissues, respectively.

Difference of FN Expression Between Young and Aging Normal Kidney Tissues
To clarify whether Fn expressions were also increased in aging kidneys, Fn protein abundances and mRNA expressions were detected. Immunofluorescent staining revealed that Fn was primarily localized in glomeruli and tubules in 3-month-old rat kidneys (Figure 5A). But in the 28-month-old rat kidneys, Fn was strikingly increased, and was observed in glomeruli, tubules, and tubulointerstitium (Figure 5A). Furthermore, Western blotting (Figure 5B) and Northern blotting (Figure 5C) demonstrated that the Fn protein abundances and the mRNA expressions were also increased with aging.



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  		Figure 5. Expression of fibronectin (Fn) in young and aging normal kidney tissues. The fluorescence intensity of Fn staining is significantly increased in the 28-month-old glomeruli and tubulointerstitium than in the 3-month-old ones (A). (B) reveals the results of Western blotting and (C) reveals the results of Northern blotting. Lane 1 indicates the 3-month-old rat kidney; lane 2 indicates the 28-month-old rat kidney. The results of semiquantitative analysis show that protein and mRNA expression of Fn is significantly more increased in the 28-month-old kidney tissues than in the young ones

 
The semiquantitative values of Fn protein and mRNA were 0.67 ± 0.10 versus 0.88 ± 0.14 (p <.01) and 0.52 ± 0.05 versus 0.62 ± 0.08 (p <.05) in young and aging rat kidneys, respectively.

Difference of Integrin-ß1 Expression Between Young and Aging Normal Kidney Tissues
Previous studies have suggested that ILK interacts with the cytoplasmic domains of ß-integrins in vitro (9). To clarify whether integrin-ß1expression changed with aging in vivo, we also investigated the integrin-ß1 protein abundances and mRNA expressions in kidneys of the 3- and 28-month-old rats by means of indirect immunofluorescent staining, Western blotting, and Northern blotting (Figure 6). Consistent with the results of ILK, expressions of integrin-ß1 protein and mRNA in kidney tissue of the 28-month-old rat group were higher than that in the 3-month-old rat group. The semiquantitative values of integrin-ß1 protein and mRNA were 0.56 ± 0.11 and 0.78 ± 0.14 (p <.05) and 0.64 ± 0.09 and 0.89 ± 0.13 (p <.05) in the 3- and the 28-month-old rat kidneys, respectively.



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  		Figure 6. Expression of integrin-ß1 in young and aging normal kidney tissues. The fluorescence intensity of integrin-ß1 in aging rat glomeruli and tubulointerstitium is higher than that of young rats (A). Western blotting (B) and Northern blotting (C) reveal that integrin-ß1 protein and mRNA expressions are significantly increased with aging. Lane 1 indicates the 3-month-old rats; lane 2 indicates the 28-month-old rats

 
The Relationship Among ILK, Integrin-ß1, and Fn in Kidney Tissues
To clarify the relationship among ILK, integrin-ß1, and Fn in kidney tissues, we carried out double immunofluorescence staining for ILK and Fn, and ILK and integrin-ß1. The results showed that ILK and Fn were only partly colocated in glomeruli and tubulointerstitium of the 3-month-old rats, but extensively colocated in that of the 28-month-old rats (Figure 7).



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  		Figure 7. Double-staining for integrin-linked kinase (ILK) and fibronectin (Fn) in young and aging kidney tissues. Green color fluorescence indicates positive ILK staining, red color fluorescence indicates positive Fn staining, orange color indicates position of ILK and Fn colocalization. Note that ILK and Fn are only partly colocalized in the 3-month-old rat glomeruli and tubulointerstitium, but Fn presents distinctly in the position of ILK cluster in the 28-month-old rat glomeruli and tubulointerstitium

 
Further, we investigated also the relationship between ILK and integrin-ß1 in kidney tissues. Regardless of whether the kidneys were from young or aging rats, ILK and integrin-ß1 were colocated in the same regions (Figure 8).



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  		Figure 8. Double-staining for integrin-linked kinase (ILK) and integrin-ß1 in young and aging kidney tissues. Green color fluorescence indicates positive ILK staining, red color fluorescence indicates positive integrin-ß1 staining, and orange indicates colocalization of ILK and integrin-ß1. Note that ILK and integrin-ß1 are colocated in same regions as both the 3-month-old and 28-month-old rat glomeruli and tubulointerstitium

 
Expression of ILK and Fn in Young and Aging Obstructed Kidney Tissues
To explore the potential role of ILK in fibrotic processes of aging kidneys under pathologic status, we investigated the expression of ILK in an aging rat model with renal interstitial fibrosis induced by UUO. The results of immunohistochemistry showed that ureteral obstruction markedly induced ILK expression in the kidney in a time-dependent fashion. ILK was increased in the obstructed kidneys on day 3, peaked on day 7, and remained at high levels on day 14 after surgery. Western blotting revealed that ILK protein abundances in aging rat kidneys were higher than that of young rats at each time point after surgery. Northern blotting also revealed a time-dependent induction of ILK mRNA in the obstructed kidneys, and the ILK expressions in the 28-month-old rat kidneys were also higher than in the 3-month-old rats at each time point (Figure 9).




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  		Figure 9. Expression of integrin-linked kinase (ILK) in young and aging obstructed kidney tissues. A, ILK immunohistochemistry staining of obstructed kidneys on day 14 after surgery in 3-month-old and 28-month-old rats. B, Graphical presentation of ILK staining positive area percentages. C and E, Western blotting and Northern blotting show ILK protein abundances and the mRNA expressions of obstructed kidney tissues in the 3-month-old and 28-month-old rats, respectively. D and F, Graphical presentation of relative ILK protein abundances normalized to actin and the mRNA expressions normalized to 28S RNA. Lane 1 represents the normal control group; lanes 2, 3, and 4 represent the unilateral ureteral obstruction (UUO) group on days 3, 7, and 14 after surgery in the 3-month-old rats, respectively. Lane 5 represents the normal control group; lanes 6, 7, and 8 represent the UUO group on days 3, 7, and 14 after surgery in the 28-month-old rats, respectively. The results show that, with the time of surgery, ILK expression was gradually raised, peaked at day 7, and maintained at high levels at day 14 after surgery. ILK protein abundances and mRNA expressions in aging rat kidneys were higher than young rat kidneys at each time point after surgery

 
The protein and mRNA expressions of Fn in the 28-month-old rat kidneys were higher than in the 3-month-old rat kidneys at each time point (Figure 10).




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  		Figure 10. Expression of fibronectin (Fn) in young and aging obstructed kidney tissues. A, Fn immunohistochemistry staining in 3-month-old and 28-month-old rat-obstructed kidneys. B, Graphical presentation of Fn positive area percentages. Northern blotting (C) shows Fn mRNA expressions of obstructed kidneys in the 3-month-old and 28-month-old rats, respectively. Lane 1 represents the normal control group; lanes 2, 3, and 4 represent the unilateral ureteral obstruction (UUO) group on days 3, 7, and 14 after surgery in the 3-month-old rats, respectively. Lane 5 represents the normal control group; lanes 6, 7, and 8 represent the UUO group on days 3, 7, and 14 after surgery in the 28-month-old rats, respectively. Graphical presentation of relative mRNA expressions normalized to 28S RNA (D). The results showed that Fn protein abundances and mRNA expressions in aging rat kidneys were higher than in young rat kidneys at each time point after surgery

 

   DISCUSSION
Top
 Abstract
 Experimental Procedures
 Results
 Discussion
References
 
ILK is an integrin-binding cytoplasmic protein that is involved in the regulation of a number of cellular processes and extracellular matrix accumulation. Their relative expressions and significance in aging kidneys, however, has not yet been reported. The present study demonstrated the relationship between ILK and aging kidneys from three lines of observation. First, from a cellular level, we found that ILK expression in glomerular mesangial cells and tubular epithelial cells of the aging rat were higher than in the young rat, and ILK expressions were positively correlated with SA-ß-gal staining and negatively correlated with cellular proliferation. Second, from the whole normal organic level, the results showed that ILK expressions were markedly increased in normal aging kidneys. Third, in a model of renal interstitial fibrosis induced by UUO, ILK expressions in obstructed kidney tissues of the aging rat were higher than that of the young at each time point after surgery, which indicates that resistance to injurious stimulation in the senescent organism was decreased. Our experimental data showed that expression of ILK was increased in cultured cells and in normal and diseased tissues of aging rat kidneys, suggesting that ILK might have a relation with the senescent process of kidneys.

ILK is an intracellular serine/threonine kinase, a multidomain focal adhesion protein, which interacts with the cytoplasmic domains of both ß1 and ß3 integrins, and phosphorylates the ß1 cytoplasmic domain in vitro (9). Recently, Goddeeris and colleagues reported that age-related declines in locomotor activity were ameliorated and mean life span was increased in Drosophila, after the gene that encoded ßPS {alpha}ß integrin in myospheroids was mutated. The data suggested that functional senescence and age-dependent mortality could be influenced by ß-integrin (25,26). As expected, the integrin-ß1 expressions were coincident with the ILK expressions in our study. Double-staining with monoclonal anti-ILK antibodies and polyclonal antiintegrin-ß1antibodies revealed that ILK and integrin-ß1 were colocalized. This study suggested that ILK might be involved in the senescent process in aging rat kidneys through mediated integrin.

Fn is a main component of the extracellular matrix. The accumulation of extracellular matrix is a characteristic of kidney senescence. However, the mechanisms of Fn deposition in aging kidneys are unclear. This study showed, along with an ILK increase, that the deposition of Fn was increased in aging kidney tissues and cells. Double immunofluorescence staining demonstrated that areas of ILK clusters accompany extensive accumulation of Fn in aging kidneys, but there is only a small amount of colocalization between Fn and ILK in young kidneys, which indicated that ILK might be related with the Fn accumulation in aging kidney. The studies have indicated that multiple integrins play critical regulative roles during the process of mediated Fn deposited into ECM (27–34). Antibodies to integrin reduced the deposition of Fn into the extracellular matrix by fibroblasts (27,29,35). However, the ability of integrins to promote Fn deposition is controlled by the integrin activation state (36). Integrin activation is regulated by integrin cytoplasmic domains. The studies suggested that intracellular proteins associated with the integrin cytoplasmic domains likely play important roles in the cellular regulation of Fn matrix assembly (37). The results presented in this article showed that the expression of ILK, integrin, and Fn were increased, and a spatial and temporal association between ILK and Fn, or between ILK and integrin, indicated that ILK promotes Fn accumulation through mediation of integrin in aging kidney. Despite our rapidly increasing knowledge of ILK promoting Fn accumulation in cultured cells and animal models (15–17,38,39), previous studies did not involve senescence.

Summary
This study explored mRNA expression and protein abundance of ILK, integrin-ß1, and Fn in primary cultured cells, normal tissues, and diseased tissues of UUO models in young and aging rat kidneys, by means of Northern blotting, Western blotting, and double-immunofluorescence staining or immunohistochemistry. The results demonstrated that the expression of ILK in primary cultured cells, normal tissues, and a UUO model of aging rat kidneys was increased compared with that of young rat kidneys, accompanied by over-expression of integrin-ß1 and Fn. Moreover, ILK expression was positively correlated with senescence-associated ß-gal positive staining, negatively correlated with cellular proliferation in kidney cells. The results indicated that ILK might be involved in the process of fibrosis and related senescence in aging kidneys.


   Acknowledgments
 
This work was supported by the National Basic Research Program of China (also called the 973 Program) (G 2000057000), National Nature Science Foundation of China (30270616 and 30300161), Postdoctoral Science Foundation of China (2003033190), and the Creative Research Group Fund of the National Natural Science Foundation of China (30121005).

C. Wu is supported by National Institutes of Health grants GM65188 and DK54639.

An abstract of this work was presented at the 2003 annual meeting of the American Society of Nephrology, San Diego, California.


   Footnotes
 
Decision Editor: Peter J. Hornsby, PhD

Received April 27, 2004

Accepted June 19, 2004


   References
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 Abstract
 Experimental Procedures
 Results
 Discussion
 References
 

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