Department of Medicine, Renal Division, Federal University of São Paulo, São Paulo, Brazil.
Exp Biol Med (Maywood). 2010 Jun;235(6):761-7. doi: 10.1258/ebm.2010.010006.
Sepsis causes impaired vascular reactivity, hypotension and acute renal failure. The ability of the Escherichia coli endotoxin (lipopolysaccharide [LPS]) to impair agonist-induced contractility in mesangial cells, which contributes to LPS-induced renal dysfunction, was evaluated. Agonist-induced intracellular calcium ([Ca(2+)]i) mobilization was analyzed using angiotensin II (AngII). The effect of LPS on the levels of the renin-angiotensin system (RAS) components and the roles of vasodilatation-inducing molecules including AT2 receptor (AT2R) and nitric oxide (NO) in the cell reactivity were also evaluated. Confluent human mesangial cells (HMCs) were stimulated with LPS (0111-B4, 100 microg/mL). AngII-induced [Ca(2+)]i mobilization was measured by fluorometric analysis using Fura-2AM in the absence and presence of an AT2R antagonist (PD123319). The mRNA and protein levels for angiotensinogen, renin, angiotensin-converting enzyme, AT1R and AT2R were analyzed by realtime reverse transcriptase-polymerase chain reaction and Western blot, respectively. NO production was measured by the chemiluminescence method in the culture media after 24, 48 and 72 h of LPS incubation. After 24 h, LPS-stimulated HMCs displayed lower basal [Ca(2+)]i and an impaired response to AngII-induced rise in [Ca(2+)]i. LPS significantly increased AT2R levels, but did not cause significant alterations of RAS components. PD123319 restored both basal and AngII-induced [Ca(2+)]i peak, suggesting an involvement of AT2R in these responses. The expected increase in NO production was significant only after 72 h of LPS incubation and it was unaffected by PD123319. Results showed that LPS reduced the reactivity of HMCs to AngII and suggest that the vasodilatation induced by AT2R is a potential mediator of this response through a pathway independent of NO.
败血症会导致血管反应受损、低血压和急性肾衰竭。本研究评估了大肠杆菌内毒素（脂多糖 [LPS]）对系膜细胞中激动剂诱导收缩性的损伤能力，这种损伤能力导致了 LPS 诱导的肾功能障碍。使用血管紧张素 II（AngII）分析激动剂诱导的细胞内钙离子 ([Ca(2+)]i) 动员。还评估了 LPS 对肾素-血管紧张素系统 (RAS) 成分水平的影响，以及包括 AT2 受体 (AT2R) 和一氧化氮 (NO) 在内的血管舒张诱导分子在细胞反应中的作用。用 LPS（0111-B4，100μg/mL）刺激汇合的人系膜细胞 (HMC)。用 Fura-2AM 通过荧光分析测量 AngII 诱导的 [Ca(2+)]i 动员，在存在和不存在 AT2R 拮抗剂（PD123319）的情况下进行。通过实时逆转录聚合酶链反应和 Western blot 分别分析血管紧张素原、肾素、血管紧张素转换酶、AT1R 和 AT2R 的 mRNA 和蛋白水平。在 LPS 孵育 24、48 和 72 h 后，通过化学发光法测量培养基中的 NO 产生。在 24 h 时，LPS 刺激的 HMC 显示出较低的基础 [Ca(2+)]i 和对 AngII 诱导的 [Ca(2+)]i 升高的反应受损。LPS 显著增加了 AT2R 水平，但没有引起 RAS 成分的显著改变。PD123319 恢复了基础和 AngII 诱导的 [Ca(2+)]i 峰值，表明 AT2R 参与了这些反应。只有在 LPS 孵育 72 h 后，NO 产生的预期增加才具有统计学意义，并且不受 PD123319 的影响。结果表明，LPS 降低了 HMC 对 AngII 的反应性，并表明 AT2R 诱导的血管舒张可能是通过独立于 NO 的途径介导这种反应的潜在介质。
