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NDT Advance Access originally published online on April 12, 2006
Nephrology Dialysis Transplantation 2006 21(7):1786-1793; doi:10.1093/ndt/gfl120
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Experimental Nephrology

Integrin-linked kinase acts as a pro-survival factor against high glucose-associated osmotic stress in human mesangial cells

Masayoshi Ohnishi, Goji Hasegawa, Masahiro Yamasaki, Hiroshi Obayashi2, Michiaki Fukui, Toshiki Nakajima, Yukiko Ichida, Hiroyuki Ohse, Shin-ichi Mogami, Toshikazu Yoshikawa1 and Naoto Nakamura

Department of Endocrinology and Metabolism and 1 Department of Inflammation and Immunology, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, 465 Kajii-cho, Hirokoji, Kawaramachi-dori, Kamikyo-ku, Kyoto, 602-8566, Japan and 2 Institute of Bio-Response Informatics, Kyoto, Japan

Correspondence and offprint requests to: Goji Hasegawa, MD, PhD, Department of Endocrinology and Metabolism, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, 465 Kajii-cho, Hirokoji, Kawaramachi-dori, Kamikyo-ku, Kyoto 602-8566, Japan. Email: goji{at}koto.kpu-m.ac.jp



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Integrin-linked kinase (ILK) is a protein that plays an important role in extracellular matrix-mediated signalling. Recent studies implicated ILK dysregulation in the development of diabetic nephropathy. However, little is known about the significance of ILK up-regulation in response to high glucose in mesangial cells.

Methods. The ILK messenger (m)RNA and protein expression in human mesangial cells were analysed with quantitative real-time polymerase chain reaction (PCR) and western blotting after exposure to either 100, 200, or 500 mg/dl glucose, or 100 mg/dl glucose + 400 mg/dl mannitol. Activation of protein Kinase B (PKB)/Akt was also determined by western blot analysis. Cells were transfected with ILK siRNA to determine the effects of ILK knockdown on PKB/Akt activation and cell death following treatment with high glucose or mannitol.

Results. High concentrations of glucose or mannitol for three days significantly up-regulated ILK mRNA and protein expression (P<0.05 vs 100 mg/dl glucose). In contrast, ILK expression in cells exposed to the same conditions for seven days was unaffected. The time course of PKB/Akt phosphorylation was similar to that of ILK protein expression. The siRNA-mediated down-regulation of ILK expression inhibited the elevation of PKB/Akt phosphorylation induced by high glucose treatment. Furthermore, the inhibition of ILK expression promoted high glucose- or mannitol-induced apoptosis.

Conclusion. The ILK may act as a pro-survival factor and play a role in protecting mesangial cells from hyperglycaemic osmotic stress.

Keywords: apoptosis; high glucose; integrin-linked kinase; mesangial cell; osmotic stress



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
Integrin-linked kinase (ILK) is an ankyrin repeat containing serine–threonine protein kinase that interacts with the cytoplasmic domains of ß-integrins and numerous cytoskeleton-associated proteins. ILK has been shown to be involved in the regulation of a number of integrin-mediated processes including cell adhesion, cell shape changes, gene expression and deposition of extracellular matrix [1]. ILK is overexpressed in a number of human malignancies [2]. Its overexpression can suppress apoptosis and promote anchorage-independent cell cycle progression through activation of the protein kinase B (PKB)/Akt kinase (phosphorylation at Ser-473) [3]. Inhibition of ILK can promote stress-induced apoptosis [4] whilst ILK overexpression may protect cells from stress-induced apoptosis [5,6], actions consistent with the role of ILK as a cell survival factor.

The ILK expression has been documented in the heart, skeletal muscle, kidney and pancreas. Recent studies implicate ILK dysregulation in the development of several chronic glomerular diseases such as congenital nephrotic syndrome, diabetic nephropathy and renal fibrosis [7–9]. Guo et al. [8] demonstrated increased ILK expression in the glomerular mesangium in human diabetic nephropathy and suggested the possible involvement of ILK in mesangial matrix expansion. However, little is known about the physiological role of ILK in mesangial cells or the significance of the ILK up-regulation in response to high glucose.

Here we show that ILK is transiently induced in glomerular mesangial cells exposed to high glucose as a result of the hyperosmotic action. We also show that ILK may act as a pro-survival factor and protect mesangial cells from hyperglycaemic/hyperosmotic stress through the activation of PKB/Akt signalling pathway.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Cell culture
Normal human glomerular mesangial cells were obtained from Cambrex Bio Science (Walkersville, MD, USA) and were cultured in mesangial cell basal medium, according to the manufacturer's instructions. Cells were grown in the presence of 100 mg/dl glucose condition until confluent and were then cultured for different periods in either 100 mg/dl, 200mg/dl or 500 mg/dl D-glucose, or a combination of 100 mg/dl D-glucose + 400 mg/dl mannitol condition. The cultures were not passaged again after exposure to the experimental conditions. This system makes the condition more akin to that in the mesangium in vivo [10]. All experiments were performed using cells between the fourth and seventh passage.

Western blot analysis
Cells grown under the conditions described above were lysed in 80 mM Tris containing 3% sodium dodecyl sulfate, 15% glycerol and protease inhibitor cocktail tablets (Roche, Mannheim, Germany), pH 6.8. For the analysis of phosphorylated PKB/Akt, 0.1 mM pervanade and 0.1 mM sodium fluoride were added to the lysis buffer. The samples were sonicated briefly to shear DNA and the protein concentration was determined using the Bio-Rad DC protein detection system (Bio-Rad, Hercules, CA, USA). About 40 µg protein extracts were then resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (pre-cast 4–20% gradient gel or 16% gel, TEFCO, Tokyo, Japan) under reducing conditions and electrotransferred to polyvinylidene difluoride (PVDF) membranes (TEFCO). Membranes were probed with one of the following primary antibodies: anti-ILK (polyclonal, Upstate Biotechnology, Lake Placid, NY, USA), anti-PKB/Akt-Pser473 (polyclonal, Upstate Biotechnology), anti-PKB/Akt (polyclonal, Upstate Biotechnology), anti-cleaved caspase 3 (polyclonal, Cell Signaling Technology, Beverly, MA, USA) and anti-actin (monoclonal, Chemicon, Temecula, CA, USA). Signals were detected using the appropriate horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit (Amersham Biosciences). Images were obtained and quantified using a Bio-Rad VersaDoc imaging system model 5000 with Bio-Rad Quantity One software. Results are expressed as a ratio (protein of interest/actin) to correct for sample loading.

Quantitative real-time PCR
Total RNA was isolated using TRIzol solution (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. Then, 1 µg of total RNA was reverse transcribed using the SuperScript RT kit from Invitrogen (Carlsbad, CA, USA). Real-time polymerase chain reaction (PCR) was performed on a fluorescence thermal cycler (Light Cycler System; Roche Diagnostics, Mannheim, Germany) using SYBR Green I as a double-strand DNA-specific dye, according to the manufacturer's protocol. In brief, the PCR amplification reaction mixtures (20 µl) contained cDNA, 0.3 µM ILK forward primer (5'-GAC ATG ACT GCC CGA ATT AG-3'), 0.3 µM reverse primer (5'-CTG AGC GTC TGT TTG TGT CT-3'), 4.0 mM MgCl2 and 1{chi} LightCycler-FastStart DNA Master SYBR Green I. In order to confirm amplification specificity, the PCR products from each primer pair were subjected to a melting curve analysis and subsequent agarose gel electrophoresis. After initial denaturation at 95°C for 10 min, reactions were cycled 40 times. Each cycle consisted of denaturation at 95°C for 10 s, primer annealing at 60°C for 10 s, and primer extension at 72°C for 10 s. Results were collected and analysed with Roche Molecular Biochemicals LightCycler Software. The software determines the crossing points of individual samples using an algorithm that identifies the first turning point of the fluorescence curve. This turning point corresponds inversely to the first maximum of the second derivative curve and correlates inversely with the log of the initial template concentration. ß-Actin cDNA was amplified using the LightCyclerTM human Primer Set (Roche Diagnostics) according to the manufacturer's protocol. Relative ILK messenger (m)RNA levels were normalized to those of ß-actin.

RNA inhibition
Human-specific ILK small interfering (si)RNA (SignalSilence ILK1 siRNA) and non-targeted negative control siRNA (SignalSilence Control siRNA) were purchased from Cell Signaling Technology (Beverly, MA, USA). Human mesangial cells at the fourth passage were grown to 50% confluence. For transfection, the siRNA – TransIT-TKO transfection reagent (Mirus, Madison, WI, USA) mixture in serum-free medium was added drop-wise onto the cell monolayer incubated in fresh serum-containing medium to yield a final concentration of 50 nM siRNA and 0.4% transfection reagent. The cells were incubated for a further 48 h and then subjected to the following experiments.

Cell death assays
Cell death was evaluated in siRNA-transfected cells cultured for two days in serum-free (0.2%) bovine serum albumin medium containing 100 mg/dl or 500 mg/dl D-glucose or a combination of 100 mg/dl D-glucose + 400 mg/dl mannitol by two independent methods: Hoechst33258 staining of nuclear DNA and the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. In addition, the activated form of caspase-3 was detected by western blot analysis in order to confirm apoptosis.

Cells were suspended in phosphate buffered saline (PBS) and stained with Hoechst 33258 (DOJINDO, Kumamoto, Japan) for 10 min and by fluorescence microscopy. Apoptotic cells were identified by nuclear condensation, shrinkage and/or fragmentation.

The WST-1 assay was performed using Premix WST-1 Cell Proliferation Assay System (Takara Bio, Shiga, Japan), according to the manufacturer's protocol. The siRNA-transfected cells grown in 96-well plates were washed with PBS, and 10 µl of the WST-1 reagent was added in 100 µl cell culture medium to each well and the cells were incubated for 2 h. Absorbance of the samples was analysed using an enzyme-linked immunosorbent assay (ELISA) reader at 450 and 690 nm. This assay was performed before and after two days of treatment with either high glucose or mannitol.

Statistical analysis
All statistical analyses were performed using the Microsoft Excel data analysis program for Kruskal–Wallis test with Dunn's multiple comparison test or Statview ver 6.0 for analysis of variance with Bonferroni's test and Mann–Whitney rank sum test. Values are expressed as means±SE.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Effect of high glucose and mannitol concentration on ILK protein and mRNA expression in human mesangial cells
Exposure of mesangial cells to 500 mg/dl glucose for three days resulted in a 1.6-fold increase in ILK protein expression (P<0.05 vs 100 mg/dl glucose, Figure 1A). Similarly, treatment of cells with the osmotic control comprising 100 mg/dl glucose + 400 mg/dl mannitol resulted in a 1.5-fold increase in ILK protein expression (P<0.05 vs 100 mg/dl glucose). In contrast, there was no difference in ILK protein expression between cells incubated for seven days in 100 mg/dl glucose, 500 mg/dl glucose or 100 mg/dl glucose + 400 mg/dl mannitol (Figure 1A).


Figure 1
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  		Fig. 1. Effect of glucose concentration on ILK expression in human mesangial cells. (A) Confluent cells were cultured for either 3 or 7 days in the presence of either 100 mg/dl (100G), 200 mg/dl (200G) or 500 mg/dl (500G) glucose or 100 mg/dl glucose + 400 mg/dl mannitol (100G + 400M). ILK protein expression was assessed by western blot analysis as described in ‘Materials and methods’ section. The bar graphs show data quantification by the densitometric scans of the ILK band from seven experiments, normalized by comparison with actin and expressed as ratio of 100G. (B) Cells were cultured for 2 days under the same condition as in the western blot analysis. ILK mRNA expression was analysed by quantitative real-time PCR as described in ‘Materials and methods’ section and normalized by that of ß-actin. The data are shown as ratio of 100G from five experiments. Values are means±SE. Kruskal–Wallis test with Dunn's multiple comparison test. *P<0.05 vs 100G. (C) Time course of increases in ILK mRNA expression. ILK mRNA expression was analysed by quantitative real-time PCR as described above and expressed as fold increase over mRNA expression at the beginning of incubation (0 h). Values are means±SE of four experiments at each time period. The increases are significant (P<0.05) at all time periods after 12 h in 500G and 100G±400M and after 96 h in 100G. Mann–Whitney rank sum test.

 
We then used quantitative real-time PCR after two days' exposure of high glucose to determine whether the increased ILK protein expression was associated with up-regulation of ILK mRNA expression. A significant increase of ILK mRNA was observed in cells incubated in 500 mg/dl glucose and 100 mg/dl glucose + 400 mg/dl mannitol compared with cells cultured in 100 mg/dl glucose (P<0.05). Furthermore, high glucose induced a dose-dependent increase in ILK mRNA (Figure 1B).

The time course of increases in ILK mRNA expression following exposure to 500 mg/dl glucose and 100 mg/dl glucose±400 mg/dl mannitol was investigated. An increase of ILK mRNA expression was detected after 6 h of incubation, with the increase in a time-dependent manner for up to 48 h of incubation, then gradually decreasing after 72 h (Figure 1C).

Effect of high glucose and mannitol concentration on PKB/Akt phosphorylation in human mesangial cells
Because previous work suggested that the PKB/Akt signalling pathway acted downstream of ILK, we analysed PKB/Akt phosphorylation at Ser-473 by western blot. The time course of PKB/Akt phosphorylation in response to the elevation of extracellular glucose or mannitol concentration was similar to that observed for ILK protein expression. Phosphorylation of PKB/Akt was significantly elevated in cells incubated for three days in 500 mg/dl glucose and 100 mg/dl glucose + 400 mg/dl mannitol compared with those in 100 mg/dl glucose (1.4-fold increase, P<0.05; Figure 2). After seven days of incubation, there were no differences in PKB/Akt phosphorylation between cells cultured under various conditions (Figure 2).


Figure 2
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  		Fig. 2. Effect of glucose concentration on PKB/Akt phosphorylation of Ser-473 in human mesangial cells. Confluent cells were cultured for either 3 or 7 days in the presence of either 100 mg/dl (100G), 200 mg/dl (200G) or 500 mg/dl (500G) glucose, or 100 mg/dl glucose + 400 mg/dl mannitol (100G + 400M). PKB/Akt phosphorylation of serine 473 was assessed by western blot analysis as described in ‘Materials and methods’ section. The bar graphs show data quantification by the densitometric scans of the Phospho-Akt Ser-473 band from seven experiments, normalized by comparing with total Akt and expressed as ratio of 100G. Values are means±SE. Kruskal–Wallis test with Dunn's multiple comparison test. *P<0.05 vs 100G.

 
High glucose concentration-induced PKB/Akt phosphorylation is abrogated by ILK inhibition in human mesangial cells
To determine whether the increased ILK protein resulting from treatment with high glucose and mannitol concentrations can functionally influence activation of PKB/Akt, cells were transfected with ILK siRNA and treated with glucose or mannitol. The transfection efficiency was determined in the same experimental conditions by counting the number of fluorescein-labelled siRNA-transfected cells by fluorescence microscopy, and was ~60%. Inhibition of ILK expression was verified by western blotting and quantitative real-time PCR (Figure 3A and B). Figure 3C demonstrates that siRNA-mediated down-regulation of ILK inhibited the high glucose-induced elevation of PKB/Akt phosphorylation.


Figure 3
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  		Fig. 3. PKB/Akt phosphorylation of serine 473 induced by hyperglycaemia was abrogated by ILK inhibition. (A) Normal human mesangial cells were transfected with ILK siRNA. Expression of ILK protein was assessed by western blot analysis 48 h after transfection. ILK siRNA inhibited endogenous ILK expression. Data shown are representative of three independent experiments. (B) ILK siRNA decreased ILK mRNA expression to 45.5% of control as assessed by real-time quantitative PCR. Data are means of three independent experiments. (C) siRNA-transfected cells were cultured for 3 days in the presence of either 100 mg/dl (100G), 500 mg/dl (500G) glucose or 100 mg/dl glucose + 400 mg/dl mannitol (100G + 400M) condition and the phosphorylation of PKB/Akt at Ser-473 was assessed by western blot analysis. The bar graphs show data quantification by the densitometric scans of the Phospho-Akt Ser-473 band from five experiments, normalized by comparing with total Akt and expressed as ratio of 100G. Knocking down ILK expression by siRNA significantly inhibited the hyperglycaemia-induced activation of PKB/Akt. Values are means±SE. Kruskal–Wallis test with Dunn's multiple comparison test. *P<0.05 vs 100G + control siRNA.

 
ILK inhibition increases human mesangial cell susceptibility to apoptosis induced by high glucose and mannitol concentrations
Following Hoechst 33258 staining, typical apoptotic cells were identified by their characteristic condensed and fragmented nuclear morphology under fluorescence microscopy. The percentage of apoptotic human mesangial cells following incubation for two days in serum-free medium with 100 mg/dl glucose was 11.8±1.2%. Exposure of control siRNA-transfected cells to high glucose or mannitol did not cause a significant increase in apoptosis (Figure 4A). However, ILK siRNA-transfected cells showed a significant increase in the level of apoptosis following exposure to high glucose (30.7±4.8%, P<0.01) or high mannitol concentrations (23.5±3.8%, P<0.05) (Figure 4A). Western blot analysis of cleaved caspase-3 clearly confirmed increased caspase-dependent apoptosis following incubation in high glucose and mannitol (Figure 4C). The WST-1 assay quantifies the viability of cells by detecting active mitochondrial enzymes and confirm the results of the Hoechst 33258 staining (Figure 4B).


Figure 4
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  		Fig. 4. Effect of ILK knockdown by siRNA on hyperglycaemia or mannitol-induced apoptosis in human mesangial cells. SiRNA-transfected cells were cultured for 2 days in serum-free medium containing either 100 mg/dl (100G), 500 mg/dl (500G) glucose or 100 mg/dl glucose + 400 mg/dl mannitol (100G + 400M). (A) Evaluation of apoptosis following staining with Hoechst 33258. Magnification 200x. Fluorescence photomicrographs of the cells treated with 500G is shown. Quantification of the experiments is shown in the graph. About 1000 cells were counted for each data point and five independent experiments were carried out. Values are means±SE. Analysis of variance with Bonferroni's test. *P<0.05 vs 100G + 400M + control siRNA. #P<0.01 vs 500G + control siRNA. (B) Evaluation of apoptosis using the WST-1 assay. At 2 days after 100G, 500G or 100G + 400M treatment, the cells were subjected to WST-1 analysis. The cleavage of WST-1 to formazan by metabolically active cells was quantified by scanning the plates at OD 450 nm. Results are means of five separate experiments containing triplicate determination for each condition. Values are means±SE. Analysis of variance with Bonferroni's test. *P<0.05 vs 500G + control siRNA, #P<0.01 vs 100G + 400M + control siRNA. (C) Apoptosis as determined by Western blot analysis of cleaved caspase-3. Representative immunoblots of the cells treated with 500G or 100G + 400M are shown. Cleaved form of caspase-3 is clearly detected in the ILK siRNA-infected cells.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
Our studies demonstrate transient up-regulation of ILK protein and mRNA expression in human glomerular mesangial cells by hyperglycaemia as a result of its hyperosmotic action. The up-regulation of ILK protein and mRNA by exposure to high concentrations of glucose have been demonstrated in rat mesangial cells and SV-40-transfected immortalized mouse podocytes [8,11]. However, the significance of the ILK up-regulation in response to high glucose has not been elucidated. The siRNA-mediated down-regulation of ILK expression suppresses the hyperglycaemia-induced phosphorylation of PKB/Akt on Ser-473 and facilitates hyperglycaemia-induced apoptosis of mesangial cells. These results indicate that hyperglycaemia-induced up-regulation of ILK could protect mesangial cells from cell death partially through activation of the PKB/Akt signalling pathway. From these studies, it can be concluded that ILK may be an important factor in the protection against hyperglycaemic-hyperosmotic stress in diabetes.

Cellular responses to elevated extracellular glucose can involve osmotic stress pathways. In addition to hyperglycaemia-induced stimulation of several interrelated pathways such as the polyol pathway, protein kinase C, hexosamine pathway, oxygen-free radicals or non-enzymatic glycation of proteins, the hyperosmotic responses of cells are thought to contribute to the pathogenesis of diabetic complications [12,13]. Hyperosmotic stress can influence a variety of important cell functions via the regulation of several ion transporters, the activity of metabolic enzymes and the transcription of certain genes. These changes include the adaptive responses to high osmolarity and survival responses (induction of anti-apoptotic signalling molecules) to protect against cell death [14,15]. Our results demonstrate a role for ILK in the prevention of mesangial cell apoptosis triggered by exposure to hyperglycaemia-associated hyperosmotic stress. They also demonstrate that PKB/Akt activation, which has been implicated as a downstream pathway of ILK-induced survival signalling [3], mediates this protective process.

Integrin–extracellular matrix interactions transduce signals that modulate the migration, proliferation, differentiation and survival of cells. It has been reported that ILK couples integrins to downstream signalling pathways involved in the suppression of apoptosis and in promoting cell cycle progression [16]. In addition to ILK, focal adhesion kinase (FAK) is an important signalling molecule in this pathway [17]. Disruption of FAK signalling results in apoptosis [18] whilst overexpression of FAK can prevent apoptosis [19]. The recent study in Swiss 3T3 cells has shown that hyperosmotic stress potently stimulates phosphorylation of FAK at Tyr-397 and Tyr-577, thereby suggesting a fundamental role for FAK in preventing apoptosis triggered by hyperosmotic stress [14]. Alternatively, study of neuronal cells demonstrated that one of the pathways of mannitol-mediated apoptosis is through dephosphorylation and degradation of FAK and Akt [20]. These results raise the possibility that the initial hyperosmotic effects, such as disruption of the actin cytoskeleton and loss of focal adhesion sites, may lead to altered integrin signalling resulting in the initiation of apoptosis and this may be the case in our study. We propose that ILK is one of the pro-survival anti-apoptotic signalling molecules which are expressed simultaneously in the face of death stimuli. Hypertonicity increases expression of genes whose protein products protect cells against hypertonicity by raising cellular levels of organic osmolytes. Accumulation of organic osmolytes reverses many effects of hypertonicity by restoring cell shrinkage and lowering intracellular ionic strength. It represents an adaptive response that apparently contributes to cell survival [21]. Therefore, it is assumed that increased ILK expression is restored following four days' exposure of high glucose and mannitol concentrations as a result of adaptation to hypertonicity. Further studies examining the potential molecular pathway of osmotic stress-induced ILK up-regulation are necessary.

The effect of hyperglycaemia on mesangial cell proliferation is controversial. Previous studies have shown that hyperglycaemia results in transient stimulation of mesangial cell proliferation followed by growth inhibition after 72 h [12]. On the other hand, it has been demonstrated that hyperglycaemia induces apoptosis in cultured mesangial cells by oxidant-dependent mechanisms [22]. However, in the present study, apoptosis was not promoted by hyperglycaemia in the absence of ILK inhibition and there were no differences in the WST-1 assay or the number of apoptotic cells between high glucose and mannitol treatments. Therefore, we may say that oxidative stress is not the main factor responsible for the initiation of hyperglycaemia-induced apoptosis in the cell culture condition employed in the present study.

Increased ILK expression has been demonstrated in the glomerular mesangium in patients with diabetic nephropathy [8]. The increase in ILK expression was associated with diffuse mesangial expansion, suggesting that the up-regulation of ILK is likely to be a relatively early event in the pathogenesis of diabetic nephropathy. In addition, it has been demonstrated that exposure of mesangial cells to high glucose increases the level of ILK and that ILK is localized to the cellular contact site to fibronectin matrix [8]. Together with the findings of other studies demonstrating the role of ILK in promoting the assembly of fibronectin matrix [23] and both fibronectin gene expression and its deposition into extracellular matrix [9], it has been suggested that ILK could be involved in mesangial matrix expansion in response to hyperglycaemia. It is also possible that ILK-stimulated assembly and production of fibronectin matrix could regulate pro-survival cell–extracellular matrix interactions [24]. Therefore, the present results demonstrating the role of ILK as a pro-survival factor against hyperglycaemic-hyperosmotic stress are consistent with the previous findings described above. Alternatively, ILK is also induced in the renal tubular epithelial cells of diabetic mice and such ILK induction is associated with epithelial to mesenchymal transition that leads to renal fibrosis in diabetic nephropathy [9].

Hyperglycaemia-induced pathological alterations in mesangial cells play a critical role in the development of diabetic nephropathy. Evaluation of the balance between complementary and opposing pathways, rather than one or two isolated components or pathways, will facilitate a better understanding of the pathogenesis of diabetic nephropathy. The present results suggest that ILK is involved in part of the complementary pathways that are activated in the event of cellular stress. Similar to our results, it has been reported that the ILK gene is induced in endothelial cells exposed to oxidized LDL, resulting in the protection of endothelial cells from apoptosis induced by oxidized LDL [5]. In addition, endothelial ILK has been suggested to play a critical role in vascular development through integrin–matrix interactions and cell survival.

Our studies indicate that ILK mRNA and protein expression is transiently induced in glomerular mesangial cells exposed to high glucose as a consequence of its hyperosmotic action. We also show that ILK could participate in protecting mesangial cells from the osmotic stress associated with hyperglycaemia via the concurrent pro-survival activation of the PKB/Akt signalling pathway. Further study of the regulation of ILK expression in the diabetic milieu should improve our understanding of the pathogenesis of diabetic nephropathy and could yield novel therapeutic targets.



   Acknowledgments
 
We thank Fumie Takenaka and Sayoko Horibe for their secretarial assistance.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 28. 1.06
Accepted in revised form: 24. 2.06


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