Acute cellular injury responses in mouse renal ischemic reperfusion injury. Mus musculus

Acute kidney injury (AKI) is associated with an abrupt loss of kidney function that results in significant morbidity and mortality. Considerable effort has focused around the identification of diagnostic biomarkers and the analysis of molecular events. Most studies have adopted organ-wide approaches that do not fully capture the interplay among different cell types in the pathophysiology of AKI. To extend our understanding of molecular and cellular events in AKI, we developed a mouse line that enables the identification of translational profiles in specific cell types by CRE recombinase-dependent activation of an eGFP-tagged L10a ribosomal protein subunit, and consequently, translating ribosome affinity purification (TRAP) of mRNA populations. By utilizing cell-type specific CRE-driver lines, in this study we identify distinct cellular responses in an ischemia reperfusion injury (IRI) model of AKI. Cell-specific translational expression profiles were uncovered 24 hours after IRI from four populations enriched for distinct anatomical and cellular subgroups: nephron, interstitial cell populations, vascular endothelium, and macrophages/monocytes by Affymetrix microarray. Overall design: A construct containing the CAGGS promoter driving eGFP-L10a, with a loxP-site flanked triple SV40 polyA cassette between promoter and eGFP-L10a cassette was targeted into the ubiquitously active Rosa26 locus. The upstream polyA cassette is designed to block activity of the downstream eGFP-L10a cassette. CRE-dependent removal of this transcriptional block activates eGFP::L10a production within the CRE-producing cell, and all of its descendants. Mice carrying the conditional eGFP-L10a allele, referred to as L10a, were maintained in a homozygous state. L10a mice were crossed to four CRE strains to activate eGFP::L10a expression in four predominantly non-overlapping cellular compartments in the kidney. A Six2-Tet-GFP::CRE allele is active exclusively within nephron progenitors; consequently, historical labeling results in eGFP::L10a expression throughout the main body of the nephron. A Foxd1-GFP::CRE allele is active in the progenitors of many of the interstitial cell lineages including those generating mesangial and non-glomerular pericytes. In addition, Foxd1 is normally expressed in podocytes. Cdh5-CRE is reported to be active throughout the vascular endothelium, and finally, Lyz2-CRE specifically labels cells of the myeloid lineage, notably macrophages, monocytes and dendritic cells. Mice carrying any CRE allele and the L10a allele are designated generically CRE-L10a. six2-L10a, foxd1-L10a, cdh5-L10a and lyz2-L10a denote specifically mice that are compound heterozygotes for the indicated CRE driver and L10a. CRE-L10a, L10a heterozygous littermates without CRE allele, C57BL/6 wild type mice were subjected to renal bilateral warm ischemia 28 minutes followed by 24-hour reperfusion when the kidney TRAP RNA and total RNA were isolated and subjected to Affymetrix microarray. Biological triplicates for each CRE-L10a line underwent no Surgery; sham Surgery and IRI treatment.

急性肾损伤（Acute kidney injury, AKI）指肾功能突发丧失，可引发严重的发病率与死亡率。学界已围绕诊断生物标志物的筛选与分子事件解析开展了大量研究，但多数研究采用全器官分析策略，无法完整捕捉AKI病理生理过程中不同细胞类型间的相互作用。
为深化对AKI分子与细胞事件的认知，我们构建了一款小鼠模型：通过CRE重组酶（CRE recombinase）依赖的eGFP标记L10a核糖体蛋白亚基激活，可实现特定细胞类型的翻译谱鉴定，进而完成mRNA群体的翻译核糖体亲和纯化（translating ribosome affinity purification, TRAP）。本研究利用细胞类型特异性CRE驱动工具鼠，在AKI的缺血再灌注损伤（ischemia reperfusion injury, IRI）模型中解析了不同细胞的特异性应答。研究通过Affymetrix微阵列，在IRI造模后24小时，从四个具有独特解剖与细胞亚群特征的富集群体中获取了细胞特异性翻译表达谱：肾单位（nephron）、间质细胞群、血管内皮细胞以及巨噬细胞/单核细胞。
整体实验设计：我们将携带CAGGS启动子驱动eGFP-L10a的构建体靶向整合至普遍表达的Rosa26基因座；该构建体在启动子与eGFP-L10a盒之间插入了由loxP位点侧翼包裹的三重SV40多聚腺苷酸（polyA）盒。该上游polyA盒可阻断下游eGFP-L10a盒的转录活性。经CRE重组酶介导切除该转录阻断序列后，CRE表达细胞及其所有子代细胞即可激活eGFP::L10a的表达。
携带条件性eGFP-L10a等位基因的小鼠（命名为L10a）以纯合子状态维持繁育。将L10a小鼠与四种CRE工具鼠杂交，以在肾脏四个基本无重叠的细胞区域中激活eGFP::L10a的表达：Six2-Tet-GFP::CRE等位基因仅在肾单位祖细胞中具有活性，因此经诱导标记后，eGFP::L10a可在肾单位主体中广泛表达；Foxd1-GFP::CRE等位基因在多种间质细胞谱系的祖细胞中具有活性，包括产生系膜细胞与非肾小球周细胞的祖细胞，此外Foxd1在正常足细胞中也有表达；Cdh5-CRE可在整个血管内皮中激活表达；而Lyz2-CRE则特异性标记髓系细胞，主要包括巨噬细胞、单核细胞与树突状细胞。
同时携带CRE等位基因与L10a等位基因的小鼠统一命名为CRE-L10a；six2-L10a、foxd1-L10a、cdh5-L10a与lyz2-L10a则特指分别携带对应CRE驱动工具与L10a等位基因的复合杂合子小鼠。将CRE-L10a小鼠、不携带CRE等位基因的L10a杂合子同窝小鼠以及C57BL/6野生型小鼠分为三组，分别接受无手术、假手术以及双侧肾脏温热缺血28分钟后再灌注24小时的IRI造模处理；随后分离各组小鼠肾脏的TRAP RNA与总RNA，进行Affymetrix微阵列检测。每一种CRE-L10a品系均设置生物学重复3次。