3.1 Acute renal injury was induced by meglumine diatrizoate
There was no statistically significant difference in basal Scr and BUN between each group. After modeling, creatinine values at 48 and 72 hours postoperatively were more than 25% higher than basal values in the group NC (0.33±0.21, 0.85±0.42, p<0.0001), which was significantly higher than that of the group N (p=0.001) (Table 5, Figure 1). The BUN level in the group NC was significantly higher after 48 hours of modeling (p=0.019) and continued to be higher after 72 hours (p=0.013)(Table 6). In the HE staining (Figure 2), compared with the kidneys of group N, which were close to the normal renal pathological manifestations, the specimens of group NC were shown severe swelling and vacuole-like degeneration of the renal tubular epithelial cell. Evenly, some of the cells underwent fragmentation and necrotic detachment that could block the tubular lumen. The renal tubules and interstitium were infiltrated with a large number of inflammatory cells, which caused a large number of tubular patterns and exudates in the tubular lumen, and even led to occlusion. Endothelial cells also became swollen and damaged, causing intracapillary coagulation and microthrombosis. It is evident that the changes in renal pathology caused by meglumine diatrizoate are mainly due to tubular damage, accompanied by changes in the tethered cells and endothelial cells. Comparison of the apoptotic index(Figure 4) by TUNEL staining (Figure 3) showed that it was significantly higher in the group NC to group N (73.60±8.64 vs. 22.37±8.73, p<0.0001), suggesting that pantethine was able to induce significant apoptosis in rat renal tubular cells.
3.2 Meglumine diatrizoate could induce apoptosis in kidney cells through ERS
The expression of GRP78 mRNA was increased in the group NC (1.250±0.186) compared to group N (1.020±0.214), but the difference was not statistically significant (p=0.0513) (Figure 5). Whlie for GADD153/CHOP and Caspase-12, the group NC had significantly higher expression compared to the N group (1.771±0.517 vs. 1.108±0.467, p=0.0426; 1.894±0.710 vs. 1.255±0.619, p=0.0472) (Figure 10, Figure 15). In the expression of proteins GRP 78, GADD153/CHOP and Caspase-12 in the apoptosis signaling pathway detected by Western Blot (Figure 6, Figure 11, Figure 16), it was clearly seen that the group NC protein image grayscales were significantly darkest among the groups (0.197±0.026, 2.539±0.270, 1.576±0.635) (Figure 7, Figure 12, Figure 17). Compared to the N group (0.118 ± 0.020, 1.582 ± 0.341, 0.249 ± 0.184), all of the three target proteins were statistically significant (p < 0.0001). Similarly as the results in the immunohistochemistry, the group NC had a significantly higher histopathological score compared to the group N (p < 0.0001) (Figure 9, Figure 14, Figure 19). The contrast agent was demonstrated to up-regulate the expression of endoplasmic reticulum-induced apoptosis signaling pathway in rat renal tubular cells.
3.3 Protective effect of drug combinations on CIAKI in rats
48 hours after modeling, the creatinine elevation rate was significantly lower in group A, P, and Q than that in group NC (p=0.049, p=0.027, p=0.026). The Scr elevation rate in group A was also significantly higher compared to group N (p=0.007), whereas that in group P and Q was similar to that of group N (p=0.159, p=0.447). For the group Q, the creatinine elevation rate was significantly lower than that of group A (p=0.026), while there was no significant difference between group P and group A. But comparing the creatinine elevation rate of group P and group Q, no significant difference was found (p=0.341). 72 hours after modeling, the level of Scr elevation in the group NC was significantly higher than that of group A (p=0.000), group P (p=0.000), and group Q (p =0.000), while there was no statistically significant difference in the level of Scr elevation between the group A, P, Q and N (Table 5, Figure 1). In the pathomorphology of rat kidney, slight swelling of renal tubular epithelial cells occurred in groups A, P and Q, only a few inflammatory cells infiltrated renal tubules and renal interstitium, a small number of tubular patterns appeared in the tubular lumen, and no microthrombus was seen. The trend of the damage in the three groups decreased sequentially(Figure 2). The number of apoptotic cells was significantly reduced in the three medicated groups compared to the group NC (p=0.0012, p<0.0001, p<0.0001), with that in groups P and Q being less than that in group A (30.95±9.61 vs. 52.95±7.67, p=0.0002; 37.61±12.13 vs. 52.95±7.67, p=0.0142). For group P and Q, there was no significant difference between the two with similar expression (p=0.3347) (Figure 4).
3.4 Effects of drug combinations on endoplasmic reticulum chaperonin expression and apoptosis in rat kidney after exposure to meglumine diatrizoate
In Real-time PCR(Figure 5, Figure 10, Figure 15), there was a trend of lower expression of the three ER chaperonin mRNA in the combination group than that in group A, but it was not statistically significant. In western blot assay for the three proteins’ expression, it can be clearly seen that the NC group has the darkest protein image grayscale among all the groups, followed by the group A, the group P and Q with the lightest image grayscale, which is comparable to the N group(Figure 6, Figure 11, Figure 16). In the semi-quantitative analysis, protein expression was significantly lower in both medicated groups than in the NC group (p<0.01). As for GADD153/CHOP, the protein expression in group P and Q was significantly lower than that in group A (p<0.0001) (Figure 12). Meanwhile, the same conclusion was obtained for group P and A for expression of GRP78 (p=0.0027) (Figure7), and there was a statistically significant difference between group Q and A in Caspase-12 (p=0.0005) (Figure 17). For the comparison between the combination groups, the expression of GADD153/CHOP and Caspase-12 was significantly lower in group Q than those in group P (p=0.0019, p=0.0244). Immunohistochemical analysis(Figure 8, Figure 13, Figure 18) showed a trend consistent with western blot, with expression decreasing roughly in the order of group NC, group A, co-administration group, and group N, in which the protein expression of all three groups with medication was significantly lower than that of group NC (p<0.05). Among them, there was a statistically significant difference between group Q and group A for GADD153/CHOP (p=0.0005) (Figure 14), and the expression of caspase-12 was significantly higher in group A compared with group P and group Q (p=0.0006, p=0.0180) (Figure 19). There was a trend that the expression of each protein in group Q was lower than that in group P, but it was not statistically significant.