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>Meprin B Activity Modulates Downstream Mediators of the Protein Kinase A Signaling Pathway in Ischemia/Reperfusion Induced Acute Kidney Injury
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Meprin B Activity Modulates Downstream Mediators of the Protein Kinase A Signaling Pathway in Ischemia/Reperfusion Induced Acute Kidney Injury
Meprins are metalloproteases that are expressed abundantly in the brush border membranes (BBM) of proximal kidney tubules. Meprins could play a role in inducing acute kidney injury (AKI) through multiple cellular and molecular mechanisms, which are currently not fully understood. The goal of the current study was to determine how meprin β expression/activity affects downstream mediators of the PKA signaling pathway in IR induced acute kidney injury. To this end, IR was induced in 12-week old wild type (WT) and meprin β knockout (βKO) mice on a C57BL/6 background by unilateral clamping of the renal artery for 27 minutes followed by 6 h of reperfusion. Blood samples were collected at 0 and 6h post-IR and centrifuged to obtain plasma for biochemical assessment of kidney function using Enzyme Linked Immunosorbent Assays (ELISA). Western blot analysis was used to determine the levels of PKA C, extracellular signal-regulated kinase (ERK), and hypoxia inducible factor 1α (HIF- 1α) in the cytosolic and nuclear-enriched protein fractions. Statistical analysis utilized 2-way ANOVA. Our data showed that meprin β enhance kidney injury as indicated by levels of NGAL and cystatin c using ELISA kits. Western blot analysis demonstrated that the expression level of Hsp27 is higher in meprin β deficient compared with wild type mice with no significant difference. The level of phospho-PKA C in cytosolic-enriched fraction has shown to be decreased in the presence of meprin following IR as demonstrated by western blot. This decreased wasn't observed in meprin β deficient mice. In contrast, the level of ERK in cytosolic and nuclear-enriched fractions is increased significantly following IR. The same pattern of ERK has been observed in the level of HIF 1α, which is increased after induction of IR. These results indicate that the low level of P-PKA C has a direct impact on the level of ERK and downstream hypoxia response genes. Elevated level of ERK and HIF 1α may thus be an indicator of kidney 2 injury. These data suggest that meprin ?, therefore, may impact IR-induced kidney injury via modulation of PKA pathway.
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机译：Meprin 是一种金属蛋白酶，在近端肾小管的刷状边界膜 （BBM） 中大量表达。Meprins 可能通过多种细胞和分子机制在诱导急性肾损伤 （AKI） 中发挥作用，目前尚不完全清楚。本研究的目的是确定 meprin β表达/活性如何影响 IR 诱导的急性肾损伤中 PKA 信号通路的下游介质。为此，在 C57BL/6 背景下，通过单侧夹闭肾动脉 27 分钟，然后再灌注 6 小时，在 C57BL/6 背景下的 12 周龄野生型 （WT） 和 meprin β 敲除 （βKO） 小鼠中诱导 IR。在 IR 后 0 小时和 6 小时收集血样并离心以获得血浆，以使用酶联免疫吸附测定 （ELISA） 进行肾功能的生化评估。Western blot 分析测定胞质和核富集蛋白组分中 PKA C 、细胞外信号调节激酶 （ERK） 和缺氧诱导因子 1α （HIF-1α） 的水平。统计分析使用 2 因子方差分析。我们的数据显示，使用 ELISA 试剂盒的 NGAL 和胱抑素 c 水平表明，meprin β 会增强肾损伤。Western blot 分析表明，与野生型小鼠相比，meprin β 缺陷小鼠中 Hsp27 的表达水平较高，无显著差异。如 western blot 所示，在 IR 后存在 meprin 的情况下，富含细胞溶质的部分的磷酸化 PKA C 水平降低。在 meprin β 缺陷小鼠中未观察到这种降低。相反，IR 后胞质和核富集级分中的 ERK 水平显着增加。在 HIF 1α 水平中观察到相同的 ERK 模式，在 IR 诱导后增加。这些结果表明，低水平 P-PKA C 对 ERK 水平和下游缺氧反应基因有直接影响。因此，ERK 和 HIF 1α 水平升高可能是肾脏 2 损伤的指标。这些数据表明，因此，meprin ？可能通过调节 PKA 通路影响 IR 诱导的肾损伤。
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North Carolina Agricultural and Technical State University.;
North Carolina Agricultural and Technical State University.;
North Carolina Agricultural and Technical State University.;
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授予单位North Carolina Agricultural and Technical State University.;North Carolina Agricultural and Technical State University.;North Carolina Agricultural and Technical State University.;
