Department of Physiology, University of Munich, Munich, Germany.
Crit Care Med. 2012 Jun;40(6):1887-95. doi: 10.1097/CCM.0b013e31824e1186.
Acute kidney injury associated with reduced urinary concentration is a frequent and severe complication during sepsis. The present study addressed the effect of endotoxemia on the functional and molecular mechanisms that determine urinary concentrating ability. Efficient urinary concentration depends on, amongst other factors, the expression of the Cl channel kidney-specific chloride channel 1 and its subunit Barttin, the urea transporter-A1, and the water channel aquaporin 2, all of which are regulated by the transcription factor TonEBP/NFAT5.
Experimental animal and cell culture model.
University laboratory.
Wistar rats and Madin-Darby canine kidney cells.
Rats were injected with lipopolysaccharide (5 mg/kg bodyweight intraperitoneal) or vehicle (phosphate-buffered saline) as control. After 24 hrs, urine, blood, and tissue samples from various kidney zones were analyzed for parameters that determine urinary concentration ability. Madin-Darby canine kidney cells were treated under isotonic or hypertonic conditions with the nitric oxide donor S-nitroso-N-acetylpenicillamine.
In rats injected with lipopolysaccharide, urine osmolality was reduced by ~40%, along with medullary induction of inducible nitric oxide synthase and a dramatic increase in urinary nitric oxide degradation products nitrite/nitrate. Concomitantly, expressions of ClC-K1, Barttin, urea transporter-A1, and aquaporin 2 were significantly lower. This was associated with the appearance of S-nitrosylated TonEBP/NFAT5, as monitored by the biotin-switch assay and immunoprecipitation, and reduced TonEBP/NFAT5 DNA binding activity in the renal inner medulla. These results were confirmed in Madin-Darby canine kidney cells transfected with a reporter construct driven by the urea transporter-A promoter, in which the nitric oxide donor S-nitroso-N-acetylpenicillamine reduces urea transporter-A reporter activity under isotonic and hypertonic conditions.
The present data demonstrate that lipopolysaccharide increases medullary nitric oxide production by iNOS induction, resulting in impairment of the transcriptional activity of TonEBP/NFAT5 by S-nitrosylation. The consequence thereof is reduced expression of TonEBP/NFAT5 target genes ClC-K1, Barttin, urea transporter-A1, and aquaporin 2 that are required for urinary concentration. Our findings may provide further insight into the molecular mechanisms underlying the urinary concentration defect in sepsis.
与尿浓缩降低相关的急性肾损伤是脓毒症的一种常见且严重的并发症。本研究旨在探讨内毒素血症对决定尿浓缩能力的功能和分子机制的影响。高效的尿浓缩依赖于多种因素，包括 Cl 通道肾脏特异性氯通道 1 及其亚基 Barttin、尿素转运蛋白 A1 和水通道 aquaporin 2 的表达，所有这些都受转录因子 TonEBP/NFAT5 的调节。
实验动物和细胞培养模型。
大学实验室。
Wistar 大鼠和 Madin-Darby 犬肾细胞。
大鼠腹腔内注射脂多糖（5mg/kg 体重）或作为对照的磷酸盐缓冲盐水。24 小时后，分析来自不同肾脏区域的尿液、血液和组织样本，以确定尿浓缩能力的相关参数。Madin-Darby 犬肾细胞在等渗或高渗条件下用一氧化氮供体 S-亚硝基-N-乙酰青霉胺处理。
在注射脂多糖的大鼠中，尿渗透压降低了约 40%，同时髓质诱导诱导型一氧化氮合酶，并显著增加了尿中一氧化氮降解产物亚硝酸盐/硝酸盐。同时，ClC-K1、Barttin、尿素转运蛋白 A1 和 aquaporin 2 的表达明显降低。这与通过生物素转移测定和免疫沉淀监测到的 TonEBP/NFAT5 的 S-亚硝基化有关，并且肾髓质中的 TonEBP/NFAT5 DNA 结合活性降低。这在转染了由尿素转运蛋白 A 启动子驱动的报告基因构建体的 Madin-Darby 犬肾细胞中得到了证实，其中一氧化氮供体 S-亚硝基-N-乙酰青霉胺在等渗和高渗条件下降低了尿素转运蛋白 A 报告基因的活性。
本研究数据表明，脂多糖通过诱导 iNOS 增加髓质中一氧化氮的产生，导致 TonEBP/NFAT5 的转录活性通过 S-亚硝基化而受损。其结果是 TonEBP/NFAT5 靶基因 ClC-K1、Barttin、尿素转运蛋白 A1 和 aquaporin 2 的表达减少，这些基因是尿浓缩所必需的。我们的发现可能为脓毒症中尿浓缩缺陷的分子机制提供进一步的见解。
