Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Division of Nephrology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Int J Mol Sci. 2024 Feb 10;25(4):2147. doi: 10.3390/ijms25042147.
Kidney transplantation is the preferred treatment for end-stage kidney disease (ESKD). However, there is a shortage of transplantable kidneys, and donor organs can be damaged by necessary cold storage (CS). Although CS improves the viability of kidneys from deceased donors, prolonged CS negatively affects transplantation outcomes. Previously, we reported that renal proteasome function decreased after rat kidneys underwent CS followed by transplantation (CS + Tx). Here, we investigated the mechanism underlying proteasome dysfunction and the role of the proteasome in kidney graft outcome using a rat model of CS + Tx. We found that the key proteasome subunits β5, α3, and Rpt6 are modified, and proteasome assembly is impaired. Specifically, we detected the modification and aggregation of Rpt6 after CS + Tx, and Rpt6 modification was reversed when renal extracts were treated with protein phosphatases. CS + Tx kidneys also displayed increased levels of nitrotyrosine, an indicator of peroxynitrite (a reactive oxygen species, ROS), compared to sham. Because the Rpt6 subunit appeared to aggregate, we investigated the effect of CS + Tx-mediated ROS (peroxynitrite) generation on renal proteasome assembly and function. We treated NRK cells with exogenous peroxynitrite and evaluated PAC1 (proteasome assembly chaperone), Rpt6, and β5. Peroxynitrite induced a dose-dependent decrease in PAC1 and β5, but Rpt6 was not affected (protein level or modification). Finally, serum creatinine increased when we inhibited the proteasome in transplanted donor rat kidneys (without CS), recapitulating the effects of CS + Tx. These findings underscore the effects of CS + Tx on renal proteasome subunit dysregulation and also highlight the significance of proteasome activity in maintaining graft function following CS + Tx.
肾移植是治疗终末期肾病（ESKD）的首选方法。然而，可供移植的肾脏短缺，捐献器官在必要的冷藏（CS）过程中可能受损。尽管 CS 提高了已故供体肾脏的活力，但长时间的 CS 会对移植结果产生负面影响。此前，我们报道了大鼠肾脏在 CS 后进行移植（CS + Tx）后，肾脏蛋白酶体功能下降。在这里，我们使用大鼠 CS + Tx 模型研究了蛋白酶体功能障碍的机制以及蛋白酶体在肾移植物结局中的作用。我们发现关键的蛋白酶体亚基β5、α3 和 Rpt6 发生了修饰，并且蛋白酶体组装受到了损害。具体来说，我们在 CS + Tx 后检测到 Rpt6 的修饰和聚集，并且当用蛋白磷酸酶处理肾提取物时，Rpt6 的修饰得到逆转。与假手术相比，CS + Tx 肾脏还显示出更高水平的硝基酪氨酸，这是过氧亚硝酸盐（一种活性氧物质，ROS）的指标。由于 Rpt6 亚基似乎聚集在一起，我们研究了 CS + Tx 介导的 ROS（过氧亚硝酸盐）生成对肾脏蛋白酶体组装和功能的影响。我们用外源性过氧亚硝酸盐处理 NRK 细胞，并评估了 PAC1（蛋白酶体组装伴侣）、Rpt6 和β5。过氧亚硝酸盐诱导 PAC1 和β5 的剂量依赖性降低，但 Rpt6 不受影响（蛋白水平或修饰）。最后，当我们抑制移植供体大鼠肾脏中的蛋白酶体（无 CS）时，血清肌酐升高，再现了 CS + Tx 的作用。这些发现强调了 CS + Tx 对肾脏蛋白酶体亚基失调的影响，并突出了蛋白酶体活性在 CS + Tx 后维持移植物功能的重要性。
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