溶血磷脂酸（Lysophosphatidic acid，LPA）是一类结构简单的生物活性脂质分子，通过与细胞膜上6个LPA受体（LPA receptor 1-6，LPAR_1-6）结合发挥广泛的生物学效应。LPA受体属于G蛋白偶联受体家族，其中LPA受体3（Lysophosphatidic acid receptor 3, LPAR₃）广泛分布于小鼠和人体的组织和器官，在生殖、心肌功能障碍、衰老等生理和病理过程中起到重要的作用。LPAR₃在小鼠肾脏中表达水平较高，但在其中发挥的生理功能还缺乏研究。肾脏的结构和功能特征使其成为对缺氧最敏感的器官之一，这一方面使肾脏进化为机体应对缺氧的效应器官，另一方面缺氧也是多种肾脏疾病的关键病理因素。肾脏应对缺氧的生物学机制研究一直是生命科学领域的经典和热点问题。已有的研究发现，LPA-LPA受体信号在多种疾病的缺氧应激过程中发挥作用，因此探究缺氧条件下LPAR₃在小鼠肾脏中发挥的生理功能及作用机制，对全面了解生物活性脂质在缺氧应激中的功能，探讨肾脏缺氧相关疾病治疗的新策略具有重要意义。

在本文中，我们以野生型（Wild type，WT）小鼠和LPAR₃敲除（LPAR₃ knock out，LPAR₃ KO）小鼠为研究对象，通过RNA测序（RNA sequencing，RNA-seq）和富集分析筛选出缺氧时肾脏LPAR₃调控的基因和生物通路，并利用分子生物学和生物化学的手段对LPAR₃在其中的作用和作用机制进行验证，取得的主要结果如下：1）LPAR₃是小鼠肾脏中表达最高的LPA受体，在缺氧条件下（8% O₂）WT小鼠肾脏中LPAR₃和催化LPA生成的关键酶Autotaxin（ATX）的表达都显著增加，这说明LPAR3信号在小鼠肾脏缺氧中可能发挥了重要功能；2）为了探究LPAR₃在小鼠肾脏缺氧中的功能，我们将WT和LPAR₃ KO小鼠置于缺氧条件12 h，RNA-seq分析两种小鼠缺氧肾脏的基因表达谱，我们发现与WT小鼠相比较，缺氧条件下LPAR₃ KO小鼠肾脏中促红细胞生成素（Erythropoietin，EPO）的表达水平和血浆中EPO的蛋白水平都显著降低，同时红细胞增殖和血红蛋白生成受阻；3）LPAR₃的靶向药物处理同样可调控缺氧肾脏的EPO表达：LPAR_1/3受体抑制剂Ki16425处理抑制缺氧条件下WT小鼠肾脏中EPO的表达，而LPAR₃选择性激动剂2S-OMPT处理则可以增强小鼠肾脏中EPO的表达；4）LPAR₃缺失造成小鼠肾脏EPO生成不足是由于缺氧时肾脏中HIF-2α累积减少；5）在体外细胞缺氧模型（1% O₂）中，靶向LPAR₃可显著抑制不同细胞系（肝癌细胞系Hep3B和肾小管上皮细胞系HK2）的HIF-2α及其靶基因EPO的表达； 6）动物组织水平和细胞水平实验显示，PI3K/Akt通路参与缺氧时LPAR₃对HIF-2α表达的调控；7）缺氧时LPAR₃的缺失造成小鼠肾小管物质运输和能量代谢能力降低，减少了细胞氧气消耗，这可能是导致HIF-2α累积减少的另一个原因。

综上所述，我们的研究发现LPAR₃在缺氧条件下小鼠肾脏EPO的生成中具有重要的作用。在小鼠全身性缺氧过程中，肾脏LPAR₃表达增加，分别通过激活下游PI3K/Akt通路，以及增加肾小管物质运输造成的氧气消耗，促进肾脏中HIF-2α累积以及EPO的表达，保证缺氧时小鼠体内的红细胞生成，在机体缺氧应激中发挥了重要功能。本文通过对LPAR₃信号在缺氧时肾脏EPO表达中的作用及机制的探究，为肾源性贫血的治疗提供了新的潜在靶点和治疗策略。

Lysophosphatidic acid (LPA) is a simple bioactive lipid mediator, exerting its extensive biological effects by binding to 6 LPA receptors (LPAR_1-6) on the cell membrane, which belong to the G protein-coupled receptor family. Among the LPA receptors, LPA receptor 3 (LPAR₃) is widely distributed in human and mouse tissues and plays a pivotal role in various physiological and pathological processes, including reproduction, cardiac dysfunction and aging. LPAR₃ is highly expressed in mouse kidneys, but its physiological function in the kidney has been poorly explored. Several structural and functional characteristics make the kidney one of the most sensitive organs to hypoxia. On the one hand, the kidney has evolved into an effector organ that cope with hypoxia. On the other hand, hypoxia has become a key pathological factor for a variety of kidney diseases. Several studies have revealed the LPA-LPAR axis played a role in hypoxia stress during several disease development. Thus, exploring the physiological functions of LPAR₃ in mouse hypoxic kidneys and the involved mechanism is significant to understand the function of bioactive lipids in hypoxic stress and to develop new strategies for the treatment of kidney hypoxia-related diseases.

In this study, high-throughput RNA sequencing and enrichment analysis were used to find the genes and biological processes regulated by LPAR₃ in mouse hypoxic kidney, and the results were later validated by molecular biology and biochemistry methods. It was found that: 1) LPAR₃ is the dominant LPA receptor in mouse kidneys, and the expression levels of LPAR₃ and Autotaxin (ATX), the major enzyme for LPA production, were increased in the kidney of WT mouse under a hypoxic condition (8% O₂), which means the important role of LPAR₃ in mouse hypoxic kidney; 2) To find the function of LPAR₃ in mouse hypoxic kidney, WT and LPAR₃ KO mice were kept under hypoxic condition for 12 h, and the kidneys were collected and analyzed by RNA-seq and found that the hypoxia induced renal EPO expression and plasma EPO protein levels in LPAR₃ KO mice was significantly lower than those in WT mice, together with decreased hematocrit and hemoglobin induced by EPO; 3) The hypoxic renal EPO production can be regulated pharmacologically by LPAR₃-targeted drugs. Under hypoxia, EPO expression in WT mice kidneys was diminished by the administration of LPAR_1/3 inhibitor Ki16425, and enhanced by 2S-OMPT, the selective agonist of LPAR₃; 4) The insufficient hypoxic renal EPO production was resulted from the reduced accumulation of HIF-2α caused by LPAR₃ deficiency; 5) In in-vitro hypoxic cell models (Hep3B cells and HK2 cells), targeting LPAR3 can also regulate HIF-2α and EPO expression; 6) Animal and cellular studies revealed that the PI3K/Akt signaling pathway was involved in the regulation of HIF-2α-EPO by LPAR₃; 7) LPAR₃ deficiency impaired renal tubular transport and energy production, resulting in lower oxygen consumption, which may be another reason for the reduced hypoxic HIF-2α accumulation.

In conclusion, we demonstrated that murine renal LPAR₃ possesses a novel function to regulate renal EPO production under hypoxia. In systemic hypoxia, the renal LPAR₃ expression is increased to promote HIF-2α accumulation and EPO induction in the kidney by activating the downstream PI3K/Akt pathway and by increasing the renal tubular transport-related oxygen consumption, which ensures the generation of red blood cells and plays an important role in the adaptation to hypoxia. The results of this study have given insights into the role and mechanism of LPAR₃ signaling in renal EPO production during hypoxia, which not only reveals a promising therapeutic target for renal anemia, but also provides new ideas for the treatment of renal anemia.