ABSTRACT
Objective:To evaluate the effect of α-ketoglutarate (AKG) on postoperative delirium (POD) in aged mice.Methods:Eighty male C57BL/6N mice, aged 18 months, weighing 30-35 g, were divided into 4 groups ( n=20 each) using a random number table method: control+ solvent group (group C), control+ AKG group (group C+ AKG), surgery+ solvent group (group S) and surgery+ AKG group (group S+ AKG). Dimethyl 2-oxoglutarate 0.6 mg/kg was injected intraperitoneally for 3 consecutive days before surgery in C+ AKG and S+ AKG groups, while the equal volume of normal saline was given in C and S groups.Exploratory laparotomy was performed under anesthesia with isoflurane to establish POD model.The behaviors of mice in each group were tested at 24 h before surgery and 6, 9 and 24 h after surgery using buried food test (the latency to eat food), open field test (total distance, latency to the center, time and freezing time spent in the center) and Y maze test (duration in the novel arm and the number of entries into the novel arm), respectively.Then the animals were sacrificed at 6 h after operation, hippocampal tissues were removed for determination of the expression of microglia-specific marker ionized calcium binding adaptor molecule-1 (Iba-1), the number of Iba-1 positive cells (using immunofluorescence staining), and the expression of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in hippocamapus (by Western blot). Results:Compared with group C, the latency to eat food at eath time point was significantly prolonged, latency to the center at 6 and 9 h after surgery was prolonged, time spent in the center at 6 and 9 h after surgery was shortened, freezing time at 6, 9 and 24 h after surgery was shortened, the number of entries into the novel arm at 6 and 9 h after surgery was decreased, duration in the novel arm at 6 h after surgery was shortened, the expression of Iba-1 was up-regulated, the number of Iba-1 positive cells was increased, and the expression of IL-1β and TNF-α in hippocampus was up-regulated in group S ( P<0.05), and no significant change was found in the behaviors indexes in group C+ AKG ( P>0.05). Compared with group S, the latency to eat food at each time point was significantly shortened, latency to the center at 9 h after surgery was shortened, time spent in the center at 6 and 9 h after surgery was prolonged, freezing time at 9 and 24 h after surgery was prolonged, the number of entries in the novel arm at 9 h after surgery was increased, the expression of Iba-1was down-regulated, the number of Iba-1 positive cells was decreased, and the expression of IL-1β and TNF-α in hippocampus was down-regulated in group S+ AKG ( P<0.05). Conclusion:AKG can alleviate POD, and the mechanism may be related to inhibiting the activation of microglia and and thus reducing inflammatory responses in aged mice.
ABSTRACT
PURPOSE: alpha-Ketoglutarate (alphaKG), a Krebs cycle intermediate, is extensively used in the kidney as a fuel substrate and as a counter anion for organic acid secretion. It is known to be taken up by the proximal tubule cells via the brush-border as well as basolateral membranes. We explored biochemical characteristics of the brush-border and basolateral alphaKG transport systems in pars convoluta and pars recta of the proximal tubule, respectively. METHODS: Brush-border and basolateral membrane vesicles (BBMV and BLMV) were isolated from rabbit renal outer cortex and outer medulla by Percoll gradient centrifugation. Vesicular uptake of alphaKG was determined by rapid Millipore filtration method using alpha-14[C]KG as a substrate. RESULTS: Both BBMV and BLMV showed a Na-gradient dependent uphill transport of alphaKG. The systems in both membranes were similarly inhibited by Li and activated by Na (Hill coefficient of 1.4). Kinetic analyses indicated that the Na-alphaKG cotransporters in the BBMV had a lower substrate affinity as compared with those in the BLMV. The transport systems in BLMVs showed a similar Km but different Vmax between the outer cortex (Km: 34 uM, Vmax: 3.3 nmol/mg protein/10s) and outer medulla (Km: 37, Vmax: 1.8). On the other hand, the systems in BBMVs were different in both Km and Vmax between the outer cortex (Km: 194, Vmax: 3.3) and outer medulla (Km: 89, Vmax: 1.7). CONCLUSION: The findings suggest that both axial and apical to basolateral heterogeneity of the Na-alphaKG cotransport system in proximal tubules may be due to a physiological adaptation to efficiently utilize alphaKG in the kidney.