(f) Xn/XO + GTP
(c) H
2O
2(d) Xn/XO
50 µµµµm
(b) GTP-pretreated
(a) control
prior to the oxidative stress induction showed the same local are representative of six independent experiments, showing similar results. The arrows represent the detached cells with necrosis.
2.
2.
2.
2. Protective effects of GTP against Xn/XOProtective effects of GTP against Xn/XOProtective effects of GTP against Xn/XOProtective effects of GTP against Xn/XO----induced oxidative induced oxidative induced oxidative induced oxidative stress
stress stress stress
The cytotoxic effects induced in the ECs were then investigated by another ROS-generating system, consisting of XO and its substrate Xn. This system enzymatically generates superoxide radicals and H2O2 during the conversion of Xn to uric acid. Figure. 2, 4 show the effects of XO activity on EC viability, as determined by FCM analysis. When the cells
were incubated with increasing XO concentrations in the presence of 250 μM Xn, a marked decrease in viability was observed. About 29% decrease in viability was observed after incubating the cells treated with 10 U/l of the enzyme. The addition of XO or Xn alone did not affect the cell viability (data not shown). Subsequently, the protective effect of GTPs against Xn/XO-induced oxidative stress in the ECs was also investigated under these experimental conditions. Pretreating the cells with 10 μg/ml GTP significantly prevented the Xn/XO-induced loss of the EC viability, indicating that the GTP acted as a biological antioxidant (Figure. 3, 4).
3. 3. vehicle-treated and GTP-treated groups at each ischemic time (Figure. 5). Mean arterial blood pressure measured during the experiments was not different between sham-operated and I/R-injured rabbits. The mortality rate induced by I/R was approximately 45%, whereas GTP administration
decreased the mortality rate to lower than 10%, except for the 120 minute ischemia group.
Figure. 5 Changes of body weights according to ischemic time. There was no difference in body weight between the vehicle-treated and GTP-treated groups at each ischemic time
vehicle GTP
4.
4.
4.
4. The effects of GTP pretreatment on renal function The effects of GTP pretreatment on renal function The effects of GTP pretreatment on renal function The effects of GTP pretreatment on renal function
When the rabbits underwent 90 minutes of ischemia, followed by 24 hours of reperfusion, creatinine levels in the blood and BUN were increased up to 3.8 ± 1.2 mg/dl and 31.3 ± 5.1mg/dl, which were 4.3 and 1.7 times higher than the levels of the control group, respectively (Figure. 6). In contrast, these phenomena were significantly (P < 0.05) prevented by GTP (200μg/kg) treatment before renal I/R, which maintained SCr and BUN levels close that of the controls.
Figure 6. The effects of green tea polyphenol (GTP) pretreatment on renal function. Serum creatinine (SCr) level
*
#
*
#
*
vehicle GTP Fig. 6A
*
#
*
vehicle GTP Fig. 6B
(A) and blood urea nitrogen (BUN) level (B) are expressed as milligrams per deciliter. The bars represent the mean ± SD.
The data was analyzed by a Tukey HSD test. The values marked with asterisks are significantly different from sham-operated groups (P < 0.05). The values marked with sharps are significantly different from GTP-treated groups (P <
0.05).
5.
5.
5.
5. The effects of GTP pretreatment on renal morphology The effects of GTP pretreatment on renal morphology The effects of GTP pretreatment on renal morphology The effects of GTP pretreatment on renal morphology A major contributor to the development and progression of I/R-induced injury is the death (deletion) of tubular epithelial cells by necrosis and apoptosis11. The control kidneys of sham-operated rabbits showed normal-looking tubular epithelial cells and glomeruli without apparent necrosis (Figure. 7A). H&E stained sections of the ischemic kidney showed the most prominent changes in the proximal tubules after 90 minutes of ischemia (Figure 7, 8). These alterations were characterized by extensive tubular epithelial necrosis, the sloughing of epithelial cells into the tubular lumen, luminal
calcification and glomerular collapse (Figure. 8). Many of the tubules were dilated, leading to increased amounts of PAS positive materials in the tubular lumen (Figure. 10). Some tubules showed the complete loss of the lining cells, whereas others showed single-cell necrosis with nuclear pyknosis and cytoplasmic eosinophilia. The changes were more pronounced in the medulla. 60 minutes of renal ischemia followed by 24 hours of reperfusion led to glomerular collapse and extensive tubular necrosis, as well (Figure. 7). Although the damage was severe in vehicle-treated or GTP-treated groups, the ischemic kidneys from rabbits that received GTP showed a much lesser degree of an ischemic injury induced epithelial damages (Figure. 7, 8) compared with the kidneys obtained from saline-treated groups. These results suggest that GTP pretreatment may result in architectural and cytologic preservation.
Histological examinations by PAS staining confirmed significant improvement in renal morphology in the GTP-treated rabbits (Figure. 9, 10). This effect was especially
remarkable in the medulla, an area that is more susceptible to oxidative damage. Glomerular collapse, an index of hypoperfusion, and platelet clots in the capillary tuft were also considerably reduced in the GTP-treated groups (Figure.
9, 10). The tubular injury score of the vehicle-administered kidneys after I/R injury was significantly (P < 0.05) increased in an ischemic time-dependent manner, while GTP pretreatment resulted in an appreciable reduction of the tubular necrosis score (Figure. 11).
Figure 7. The renal histology of sham-operated kidney (A), vehicle-treated kidney (B, C) and GTP-treated kidney (D, E) after 60 minutes(B, D) and 90 minutes(C, E) of ischemia, followed by 24 hours of reperfusion. The photographs shown in this figure are representative of 6 independent rabbits showing similar results. The arrows represent the glomerular collapse (Hematoxylin-eosin stain, Original magnification × 100).
60 min 90 min
(A)
(B)
(D)
(C)
(E)
Figure 8. The renal histology of vehicle-treated kidney (A) and GTP-treated kidney (B) after 90 minutes of ischemia, followed by 24 hours of reperfusion. Collapsed glomeruli, luminal calcification, and sloughing of epithelial cells into the tubular lumen were observed in the vehicle-treated kidney (A). But, GTP-treated kidney (B) demonstrated intact tubular lumen and glomerulus. (H & E stain, Original magnification × 100).
A
B
Figure 9. The PAS stain of sham-operated kidney (A), vehicle-treated kidney (B, C) and GTP-vehicle-treated kidney (D, E) after 60 minutes(B, D) and 90 minutes(C, E) of ischemia, followed by 24 hours of reperfusion. The arrows represent intraluminal calcification and the asterisks (*) in (B) represent PAS positive materials in the tubular lumens (PAS stain, Original magnification ×
(B)
(D) (A)
* * *
*
60 min 90 min
(E)
(C)
Figure 10. The renal histology of vehicle-treated kidney (A) and GTP-treated kidney (B) with PAS stain after 90 minutes of ischemia, followed by 24 hours of reperfusion. Collapsed glomeruli, luminal calcification, and disfigurations of basement membrane on the tubules (A). But, GTP-treated kidney (B) was observed some intact basement membrane and glomeruli.
A
B
Figure 11. The tubular injury score of vehicle-treated and GTP-treated kidneys in rabbits after IR. The bars represent the mean ± SD. The data was analyzed by a Tukey HSD test.
The values marked with asterisks are significantly different from sham-operated groups (P < 0.05). The values marked with sharps are significantly different from GTP-treated groups (P < 0.05).
IV.
IV.
IV.
IV. DISCUSSION DISCUSSION DISCUSSION DISCUSSION 0