肾脏疾病的复杂发病机制与肾脏固有细胞的多样性密切相关。在这项研究中,采用单细胞转录组测序技术对正常对照大鼠和慢性肾脏病(CKD)大鼠的血液和肾组织细胞进行测序和分析,重点研究关键细胞群和功能富集,探讨CKD的发病机制。采用油红O染色和酶联免疫吸附试验(ELISA)检测脂滴和游离脂肪酸(FFA)。定量实时聚合酶链反应(RT-PCR),采用westernblot(WB)方法验证组织中差异基因羟基酸氧化酶2(HAO2)和脂肪酸代谢过程,确保单细胞测序结果的可靠性。我们成功建立了CKD大鼠血液和肾脏组织的单细胞转录组图谱,它们被注释成14个细胞亚群(MPCs,PT,Tc,DCT,B-IC,A-IC,CNT,ALOH,BC,Neu,远藤,Pla,NKT,Baso)根据标记基因,而整合的大鼠单细胞图谱显示CKD组MPCs和PTs明显升高和降低,分别。功能分析发现PT细胞中代谢相关途径的广泛富集,包括脂肪酸代谢过程,细胞氨基酸代谢过程和前体代谢物和能量的产生。免疫组织化学实验确定差异基因HAO2定位于肾小管中,与对照组相比,CKD组的表达明显减少,油红O染色显示CKD组脂滴增多,HAO2过表达后,脂滴受到抑制。ELISA检测显示,CKD组ATP含量降低,FFA升高。此外,与OE-NC相比,OE-HAO2组细胞的线粒体膜电位显着增加。酰基辅酶A氧化酶1(ACOX1),过氧化物酶体增殖物激活受体α(PPARα),过氧化物酶体增殖物激活受体γ-辅激活因子1-α(PGC1α)在CKD组降低,HAO2过表达后基因和蛋白质增加,AMP激活的蛋白激酶(AMPK)磷酸化蛋白增加,乙酰辅酶A羧化酶(ACC)磷酸化蛋白减少,相反。因此,HAO2可能是CKD脂肪酸代谢过程的重要调节因子,高表达HAO2可通过促进脂肪酸氧化(FAO)途径增强脂肪酸代谢。
The complex pathogenesis of kidney disease is closely related to the diversity of kidney intrinsic cells. In this study, single-cell transcriptome sequencing technology was used to sequence and analyze blood and kidney tissue cells in normal control rats and rats with chronic kidney disease (CKD), focusing on key cell populations and functional enrichment to explore the pathogenesis of CKD. Oil red O staining and enzyme-linked immunosorbent assay (ELISA) were used to detect lipid droplets and free fatty acid (FFA). Quantitative real-time polymerase chain reaction (RT-PCR), western blot (WB) were used to verify the differential gene hydroxyacid oxidase 2 (HAO2) and fatty acid metabolic process in tissue to ensure the reliability of single-cell sequencing results. We successfully established a single-cell transcriptome atlas of blood and kidney tissue in rats with CKD, which were annotated into 14 cell subsets (MPCs, PT, Tc, DCT, B-IC, A-IC, CNT, ALOH, BC, Neu, Endo, Pla, NKT, Baso) according to marker gene, and the integrated single-cell atlas of rats showed a significant increase and decrease of MPCs and PTs in the CKD group, respectively. Functional analysis found extensive enrichment of metabolic-related pathways in PT cells, includes fatty acid metabolic process, cellular amino acid metabolic process and generation of precursor metabolites and energy. Immunohistochemical experiments determined that the differential gene HAO2 was localized in the renal tubules, and its expression was significantly reduced in CKD group compared with control, and oil red O staining showed that lipid droplets increased in the CKD group, after overexpression of HAO2 the lipid droplets was inhibited. ELISA assay showed that ATP content decreased in the CKD group and FFA increased in the CKD group. Moreover, the mitochondrial membrane potential of the cells in the OE-HAO2 group was significantly increased compared with OE-NC. The acyl-CoA oxidase 1(ACOX1), peroxisome proliferator-activated receptor alpha (PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were decreased in the CKD group, while genes and proteins were increased after overexpression of HAO2, and the AMP-activated protein kinase (AMPK) phosphorylated proteins were increased, the acetyl-CoA carboxylase (ACC) phosphorylated proteins were decreased, reversely. Therefore, HAO2 may be an important regulator of fatty acid metabolic processes in CKD, and overexpression of HAO2 can enhance fatty acid metabolism by promoting fatty acid oxidation (FAO) pathway.