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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 28, 2003 as doi:10.1096/fj.02-0130fje. |
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Department of Experimental Medicine and Nephrology, William Harvey Research Institute, St. Bartholomew’s and Royal London School of Medicine and Dentistry, Queen Mary, University of London, UK
2Correspondence: Department of Experimental Medicine and Nephrology, Suite 22, Dominion House, 59 Bartholomew Close, West Smithfield, London EC1A 7BE, UK. E-mail: d.a.allen{at}qmul.ac.uk
SPECIFIC AIMS
The development of diabetic nephropathy is associated with increased peroxynitrite (ONOO–) generation but the mechanisms of high glucose-induced tissue injury are poorly understood. To better understand the role of ONOO– in diabetic nephropathy, we evaluated the extent of oxidative stress and apoptosis occurring in proximal tubular epithelial cells after exposure to high glucose and attempted to further define mediators of the injury by the use of inhibitors of ONOO– generation.
PRINCIPAL FINDINGS
1. High glucose causes peroxynitrite generation in LLC-PK1 cells
The porcine proximal tubular epithelial cell line (LLC-PK1) was used to assess the effect of high glucose on cellular injury. Subconfluent monolayers of the LLC-PK1 cell line were exposed to two different glucose concentrations (5 or 25 mM) for 24 h. Oxidative stress was monitored using the cell-permeable fluorogenic probe 2,3-dichlorofluorscein diacetate (DCF-DA). The dichlorofluorescein (DCF) then reacts with peroxides (including ONOO–) to generate the fluorescent molecule DCF. Cells exposed to low glucose (5 mM D-glucose plus 20 mM L-glucose to ensure isosmolarity) generated a low-level fluorescence, indicating there is some peroxide generation in controls. However, there was a 70% increase in fluorescence when LLC-PK1 cells were exposed to 25 mM glucose for 24 h (n=9, P<0.02). When the experiment was repeated in the presence of 10 µM ebselen, the high glucose-induced DCF fluorescence was much reduced. Ebselen is a seleno-methionine-based compound known to be an effective scavenger of ONOO– in biological systems. We concluded from these experiments that high glucose causes the generation of ONOO– in proximal tubular cells. This was confirmed by experiments using 100 µM 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, an analog of vitamin E) in which DCF fluorescence was also reduced. The use of another probe for peroxynitrite (dihydrorhodamine 123) yielded similar results.
Nitrite concentrations were measured in supernatants taken from cells exposed to high glucose for 24 h and were significantly higher than controls (n=4, P<0.05). Nitrite concentrations were also increased 24 h after direct addition of ONOO– to LLC-PK1 cells. Ebselen reduced the high glucose-induced increase in nitrite concentration (n=4, P<0.05) whereas a nitric oxide inhibitor (N-(3-(aminomethyl)benzyl)acetamidine, 1400W) had no effect. Western blots showed increased nitrotyrosine staining on several proteins, providing further evidence of increased peroxynitrite generation in high glucose treated LLC-PK1 cells.
2. High glucose-induced peroxynitrite generation leads to apoptotic cell death in LLC-PK1 cells
To evaluate the effect of ONOO– generation on cell death in LLC-PK1 cells, we used a sensitive DNA fragmentation ELISA to assess apoptosis. LLC-PK1 cells were exposed to high ambient glucose concentrations for up to 48 h and cytosolic extracts were prepared using PBS (pH 7.4) containing 10 µM digitonin. DNA fragmentation was assessed with reference to control cells cultured in 5 mM D-glucose plus 20 mM L-glucose as before. Cells exposed to high glucose for 48 h showed 2.5-fold increased DNA fragmentation compared with controls (n=4, P<0.02). As a second marker of apoptosis, annexin V staining was measured on intact LLC-PK1 cells and found to be higher in cells treated with high glucose than in controls (6.6±0.7% vs 3.5±0.3%, n=3, P<0.05). ONOO– generation occurred maximally at 24 h after exposure to high glucose; although DNA fragmentation was evident 24 h after initial exposure to high glucose, it reached a maximum at 48 h. The data suggest that high glucose-induced ONOO– generation occurred before apoptosis in LLC-PK1 cells. To confirm this hypothesis, DNA fragmentation was assessed in LLC-PK1 cells exposed to high glucose in the presence of ebselen. Ebselen prevented an increase in DNA fragmentation in LLC-PK1 cells exposed to high glucose (n=6, P<0.05, Fig. 1
A).
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3. The role of caspases in high glucose-induced LLC-PK1 cell apoptosis
The experiments described above identified apoptotic cell death in high glucose-treated LLC-PK1 cells. Based on this, we decided to investigate the role of caspases in this process. We measured the activity of caspases -3, -8, and -9 in LLC-PK1 cytosolic lysates. Cells were incubated for up to 48 h in media containing 5 mM or 25 mM D-glucose with or without ebselen. Caspase-3 activity was threefold higher in cells grown in 25 mM D-glucose for 48 h than in controls (n=9, P<0.05). The high glucose-induced increase in caspase-3 activity was reduced by 30% in the presence of ebselen (n=3, P<0.05, Fig. 1B
), implying that ONOO– causes caspase-3 activation and subsequent apoptosis in high glucose-treated LLC-PK1 cells. In this model, caspase-8 activity was increased but did not reach significance compared with controls. However, caspase-9 activity was significantly higher in LLC-PK1 cells exposed to high glucose at 24 h (n=3, P<0.01), and this activity was reduced by treatment with ebselen (n=3, P<0.05). To further define the role of caspases in high glucose-induced proximal tubular cell apoptosis, we used three different cell-permeable caspase inhibitors: an inhibitor of caspase-3 (Ac-DEVD-CHO), an inhibitor of caspase-8 (Ac-IETD-CHO), and a pan-caspase inhibitor, Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-D-DCB). DNA fragmentation and caspase-3 activity were measured after 48 h of exposure to high glucose with or without the inhibitors. All three inhibitors completely attenuated caspase-3 activity (n=6, P<0.001 for all inhibitors, Fig. 2
A) and prevented the high glucose-induced DNA fragmentation (Ac-DEVD-CHO, n=8, P<0.05; Ac-IETD-CHO, n=4, P<0.05; Z-Asp-DCB, n=8, P<0.005, Fig. 2B
). Z-Asp-DCB was the most effective inhibitor. The mechanism by which high glucose-induced ROS activate caspases is not clear. We examined the expression of X-linked inhibitor of apoptosis (XIAP) in cells exposed to high glucose for 24 or 48 h. XIAP inhibits caspases either by preventing cleavage of the pro-caspase or by direct inhibition of the active caspase subunit. At both 24 and 48 h, XIAP expression was much reduced in cells exposed to 25 mM D-glucose compared with controls.
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CONCLUSIONS AND SIGNIFICANCE
The damaging effects of high ambient glucose concentrations on various cell types have been known for some time. Nevertheless, there remains considerable doubt about the cellular mediators of the injury. Here we have identified for the first time the caspase cascade—in particular, caspase-3—as a final common mediator of high glucose-induced proximal tubular apoptosis. We were able to prevent apoptosis using three different caspase inhibitors, demonstrating that multiple caspases contribute to activate caspase-3. The role of caspase-8 in high glucose-induced apoptosis in PTECs must be interpreted with caution. The data demonstrating that XIAP is down-regulated provides an attractive hypothesis for the mechanism of high glucose-induced PTEC apoptosis and is currently under investigation in our laboratory. It is possible that ROS such as ONOO– could initiate these processes (Fig. 3
).
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We showed there is increased oxidative stress in the form of peroxynitrite in high glucose treated LLC-PK1 cells. Peroxynitrite is a powerful oxidant that can perform one- and two-electron oxidations and react with numerous biologically important molecules such as thiols, amines, lipids, and proteins. At physiological pH, 
20% of ONOO– is protonated to peroxynitrous acid. This is relevant since this form of ONOO– can react with cytochrome c. This reaction may lead to activation of apoptotic mechanisms involving mitochondrial proteins. Due to the abundance of CO2 in biological systems, a major reaction of ONOO– is with CO2 or bicarbonate. This reaction yields nitrosoperoxycarbonate, which can nitrosylate proteins as well as carry out one- and two-electron oxidations. Two additional ROS are generated by decomposition of the nitrocarbonate anion—nitrogen dioxide and the carbonate anion—further enhancing oxidative stress.
In the present study we were able to prevent the damaging effects of high glucose with ebselen. Due to its high rate constant for the reaction with ONOO– (1.6x106 M–1s–1), ebselen is one of a small number of compounds that can compete with CO2 for ONOO–.
The source of ONOO– in high glucose-treated LLC-PK1 cells remains unclear. Since ONOO– is formed by the diffusion controlled reaction between nitric oxide and superoxide, we attempted to inhibit ONOO– formation with a NOS inhibitor (1400W). However, 1400W did not reduce DCF fluorescence nor did it significantly reduce nitric oxide generation (measured by detecting nitrite in supernatants). One possible explanation for this result is that nitrite can react with hydrogen peroxide to form peroxynitrous acid. The generation of hydrogen peroxide is known to occur in various diseases, including diabetes, and could provide an indirect route for ONOO– generation. Despite the uncertainty regarding the source of ONOO– in high glucose-treated LLC-PK1 cells, our data provide clear evidence of peroxynitrite-induced apoptosis.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0130fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0130fje 
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