比较一种新型离体器官保存液(A液)与威斯康星大学保存液(UW液)、高渗枸橼酸盐腺嘌呤液(HCA液)对大鼠离体肾脏的保存效能。

通过测定研制的A液离子浓度、pH值和渗透压，验证其理化性质与稳定性。通过CCK-8实验，探究A液对人胚肾上皮细胞系(293T)和肾小管上皮细胞(HK-2)的细胞活力及凋亡影响。构建大鼠供肾模型，采用随机数字表法将15只大鼠30个肾脏随机分为UW液组、HCA液组和A液组，每组各10个。将离体肾脏置于相应保存液中，分别于0、6、12、24、48、72 h收集肾组织标本。通过HE染色评估组织病理损伤，比色法测定ATP酶活性及丙二醛(MDA)含量，TUNEL染色定量分析细胞凋亡情况，蛋白质印迹法检测凋亡相关蛋白(Caspase-3、Cleaved Caspase-3、Bax、Bcl-2)及炎症因子(HO-1、TNF-α)表达。所有数据均经过至少3次重复实验获得。计量资料多组间比较采用方差分析，进一步两两比较使用Dunnett检验，P<0.05为差异有统计学意义。

体外实验显示：A液组细胞活力高于UW液组、低于HCA液组(P均<0.05)。离体大鼠肾脏保存实验显示：组织病理学方面，保存48 h时，A液组肾小管空泡化程度低于HCA液组(P<0.05)，且与UW液组相比差异无统计学意义(P>0.05)。氧化应激指标中，保存48、72 h时，A液组ATP酶活性均高于UW液组和HCA液组，差异均有统计学意义(P均<0.05)。A液组MDA累积速率低于HCA液组和UW液组，48、72 h MDA含量(0.27%、0.28%)均低于HCA液组(0.42%、0.42%)，差异均有统计学意义(P均<0.05)。细胞凋亡方面，保存24 h后，A液组TUNEL凋亡指数低于HCA液组，Bcl-2/Bax比值高于HCA液组，差异均有统计学意义(P均<0.05)；Cleaved Caspase-3蛋白相对表达量低于UW液组和HCA液组(P<0.05)。炎症因子方面，A液组TNF-α表达水平低于HCA液组(P<0.05)，HO-1表达水平低于UW液组和HCA液组(P<0.05)。

新型器官保存液在48 h内对大鼠肾脏展现出与市售经典保存液相当或更优的保护潜力。在72 h延长保存条件下，新型器官保存液可通过维持ATP酶活性、降低MDA含量并调控凋亡相关通路，具有出色的肾脏保护效能。

To compare the preservation efficacy of a novel ex vivo organ preservation solution (Solution A) was compared with that of University of Wisconsin solution (UW solution) and hypertonic citrate adenine solution (HCA solution) on isolated rat kidneys.

The physicochemical properties and stability of the novel ex vivo organ preservation solution developed at our center were validated by measuring its ion concentration, pH value, and osmolality. The cytotoxicity and apoptosis effects of Solution A on human embryonic renal epithelial cell line (293T) and renal tubular epithelial cells (HK-2) were investigated using the CCK-8 assay. A rat kidney donor model was established. A total of 30 kidneys from 15 rats were randomly divided into three groups (UW solution group, HCA solution group, and Solution A group) using a random number table method, with 10 kidneys in each group. The isolated kidneys were placed in the corresponding preservation solutions, and renal tissue samples were collected at 0, 6, 12, 24, 48, and 72 hours. Histopathological damage was assessed by HE staining, ATPase activity and malondialdehyde (MDA) content were determined using colorimetric assays. Apoptosis was quantitatively analyzed by TUNEL staining, and the expression of apoptosis-related proteins (Caspase-3, Cleaved Caspase-3, Bax, Bcl-2) as well as inflammatory factors (HO-1, TNF-α) was detected by Western blotting. All experimental data were obtained from at least three indepent replicates. Comparisons of experimental data among multiple groups were performed using analysis of variance (ANOVA), followed by pairwise comparisons using Dunnett′s test. A P value <0.05 was considered statistically significant.

In vitro experiments demonstrated that the cell viability of the Solution A group was significantly higher than that of the UW solution group and lower than that of the HCA solution group (all P<0.05). In vitro rat kidney preservation experiments showed that at 48 hours of preservation, the degree of tubular vacuolization in the Solution A group was significantly lower than that in the HCA solution group (P<0.05), but no statistically significant difference was observed compared to the UW solution group (P>0.05). For oxidative stress indicators, the ATPase activity of the Solution A group was significantly higher than that of the UW solution and HCA solution groups at 48 and 72 hours of preservation (all P<0.05). In addition, the MDA accumulation rate in the Solution A group was lower than that in the HCA and UW solution groups, and the MDA content at 48 and 72 hours (0.27%, 0.28%) was significantly lower than that of the HCA solution group (0.42%, 0.42%) (all P<0.05). After 24 hours of preservation, the Solution A group exhibited a significantly lower TUNEL apoptosis index and a higher Bcl-2/Bax ratio compared with the HCA solution group (all P<0.05), as well as a lower relative expression level of Cleaved Caspase-3 protein compared with both the UW and HCA solution groups (P<0.05). In terms of inflammatory factors, the TNF-α expression level in the Solution A group was significantly lower than that in the HCA solution group (P<0.05), and the HO-1 expression level was significantly lower than that in both the UW and HCA solution groups (P<0.05).

The novel organ preservation solution exhibited renal protective potential comparable to, or even better than, that of commercially available classic preservation solutions within 48 hours in rats. Under prolonged preservation conditions up to 72 hours, the novel solution further exhibited excellent renal protective efficacy by maintaining ATPase activity, reducing MDA content, and regulating apoptosis-related pathways.