Laboratorio de Fisiología Integrativa y Molecular, Programa de Fisiología, Centro de Investigación e Innovación Biomédica, Universidad de los Andes, Santiago, Chile.
Escuela de Medicina, Facultad de Medicina, Universidad de los Andes, Santiago, Chile.
Cell Physiol Biochem. 2022 Nov 16;56(6):613-628. doi: 10.33594/000000586.
BACKGROUND/AIMS: The renal inflammatory response and kidney regeneration in ischemia-reperfusion injury (IRI) are associated with Toll-like receptor 4 (TLR4). Here we study the role of TLR4 during IRI in the renal cortex and medulla separately, using wild-type (TLR4-WT) and Knockout (TLR4-KO) TLR4 mice.
We used 30 minutes of bilateral renal ischemia, followed by 48 hours of reperfusion in C57BL/6 mice. We measured the expression of elements associated with kidney injury, inflammation, macrophage polarization, mesenchymal transition, and proteostasis in the renal cortex and medulla by qRT-PCR and Western blot. In addition, we studied kidney morphology by H/E and PAS.
Renal ischemia (30min) and reperfusion (48hrs) induced the mRNA and protein of TLR4 in the renal cortex. In addition, Serum Creatinine (SCr), blood urea nitrogen (BUN), Neutrophil gelatinase-associated lipocalin (NGAL), and acute tubular necrosis (ATN) were increased in TLR4-WT by IRI. Interestingly, the SCr and BUN had normal levels in TLR-KO during IRI. However, ATN and high levels of NGAL were present in the kidneys of TLR4-KO mice. The pro-inflammatory (IL-6 and TNF-α) and anti-inflammatory (Foxp3 and IL-10) markers increased by IRI only in the cortex of TLR4-WT but not in TLR4-KO mice. Furthermore, the M1 (CD38 and Frp2) and M2 (Arg-I, Erg-2, and c-Myc) macrophage markers increased by IRI only in the cortex of TLR4-WT. The TLR4-KO blunted the IRI-upregulation of M1 but not the M2 macrophage polarization. Vimentin increased in the renal cortex and medulla of TLR4-WT animals but not in the cortex of TLR4-KO mice. In addition, iNOS and clusterin were increased by IRI only in the cortex of TLR4-WT, and the absence of TLR4 inhibited only clusterin upregulation. Finally, Hsp27 and Hsp70 protein levels increased by IRI in the cortex and medulla of TLR4-WT and TRL4-KO lost the IRI-upregulation of Hsp70. In summary, TLR4 participates in renal ischemia and reperfusion through pro-inflammatory and anti-inflammatory responses inducing impaired kidney function (SCr and BUN). However, the IRI-upregulation of M2 macrophage markers (cortex), iNOS (cortex), IL-6 (medulla), vimentin (medulla), and Hsp27 (cortex and medulla) were independent of TLR4.
The TLR4 inactivation during IRI prevented the loss of renal function due to the inactivation of inflammation response, avoiding M1 and preserving the M2 macrophage polarization in the renal cortex.
背景/目的：肾缺血再灌注损伤（IRI）中的肾炎症反应和肾脏再生与 Toll 样受体 4（TLR4）有关。在这里，我们使用野生型（TLR4-WT）和敲除型（TLR4-KO）TLR4 小鼠分别研究 TLR4 在肾皮质和髓质中的作用。
我们使用双侧肾缺血 30 分钟，然后再灌注 48 小时，在 C57BL/6 小鼠中进行。我们通过 qRT-PCR 和 Western blot 测定与肾损伤、炎症、巨噬细胞极化、间充质转化和蛋白质稳态相关的元素在肾皮质和髓质中的表达。此外，我们通过 H/E 和 PAS 研究了肾脏形态。
肾缺血（30min）和再灌注（48hrs）诱导了肾皮质中 TLR4 的 mRNA 和蛋白表达。此外，TLR4-WT 中的血清肌酐（SCr）、血尿素氮（BUN）、中性粒细胞明胶酶相关脂质运载蛋白（NGAL）和急性肾小管坏死（ATN）在 IRI 后增加。有趣的是，在 TLR-KO 中，SCr 和 BUN 在 IRI 期间水平正常。然而，在 TLR4-KO 小鼠的肾脏中存在 ATN 和高水平的 NGAL。仅在 TLR4-WT 的皮质中，促炎（IL-6 和 TNF-α）和抗炎（Foxp3 和 IL-10）标志物因 IRI 而增加，而在 TLR4-KO 小鼠中则没有。此外，仅在 TLR4-WT 的皮质中，M1（CD38 和 Frp2）和 M2（Arg-I、erg-2 和 c-Myc）巨噬细胞标志物因 IRI 而增加。TLR4-KO 减弱了 IRI 对 M1 的上调，但对 M2 巨噬细胞极化没有影响。波形蛋白在 TLR4-WT 动物的肾皮质和髓质中增加，但在 TLR4-KO 动物的皮质中没有增加。此外，iNOS 和 clusterin 仅在 TLR4-WT 的皮质中因 IRI 而增加，而 TLR4 的缺失仅抑制 clusterin 的上调。最后，Hsp27 和 Hsp70 蛋白水平在 TLR4-WT 的皮质和髓质中因 IRI 而增加，而 TLR4-KO 失去了 IRI 对 Hsp70 的上调。综上所述，TLR4 通过促炎和抗炎反应参与肾缺血再灌注，导致肾功能受损（SCr 和 BUN）。然而，M2 巨噬细胞标志物（皮质）、iNOS（皮质）、IL-6（髓质）、波形蛋白（髓质）和 Hsp27（皮质和髓质）的 IRI 上调与 TLR4 无关。
在 IRI 期间，TLR4 的失活可防止因炎症反应失活而导致的肾功能丧失，避免 M1 并在肾皮质中保留 M2 巨噬细胞极化。
