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Abstract：
This study aimed to identify an orcl1 mutation in a patient with Dent-2 Disease and investigate the underlying mechanisms.
The ocrl1 mutation was identified through exome sequencing. Knockdown of orcl1 and overexpression of the orcl1 mutant were performed in HK-2 and MPC5 cells to study its function, while flow cytometry measured reactive oxygen species (ROS), phosphatidylserine levels, and cell apoptosis. Scanning electron microscopy observed crystal adhesion, while transmission electron microscopy examined kidney tissue pathology. Laser scanning confocal microscopy was used to examine endocytosis, and immunohistochemical and immunofluorescence assays detected protein expression. Additionally, podocyte-specific orcl1 knockout mice were generated to investigate the role of orcl1 in vivo.
We identified a mutation resulting in the replacement of Histidine with Arginine at position 318 (R318H) in ocrl1 in the proband. orcl1 was widely expressed in the kidney. In vitro experiments showed that knockdown of orcl1 and overexpression of ocrl1 mutant increased ROS, phosphatidylserine exocytosis, crystal adhesion, and cell apoptosis in HK-2 cells. Knockdown of orcl1 in podocytes reduced endocytosis and disrupted the cell cycle while increasing cell migration. In vivo studies in mice showed that conditional deletion of orcl1 in podocytes caused glomerular dysfunction, including proteinuria and fibrosis.
This study identified an R318H mutation in orcl1 in a patient with Dent-2 Disease. This mutation may contribute to renal injury by promoting ROS production and inducing cell apoptosis in tubular cells, while disrupting endocytosis and the cell cycle, and promoting cell migration of podocytes. Video Abstract.
The ocrl1 mutation was identified through exome sequencing. Knockdown of orcl1 and overexpression of the orcl1 mutant were performed in HK-2 and MPC5 cells to study its function, while flow cytometry measured reactive oxygen species (ROS), phosphatidylserine levels, and cell apoptosis. Scanning electron microscopy observed crystal adhesion, while transmission electron microscopy examined kidney tissue pathology. Laser scanning confocal microscopy was used to examine endocytosis, and immunohistochemical and immunofluorescence assays detected protein expression. Additionally, podocyte-specific orcl1 knockout mice were generated to investigate the role of orcl1 in vivo.
We identified a mutation resulting in the replacement of Histidine with Arginine at position 318 (R318H) in ocrl1 in the proband. orcl1 was widely expressed in the kidney. In vitro experiments showed that knockdown of orcl1 and overexpression of ocrl1 mutant increased ROS, phosphatidylserine exocytosis, crystal adhesion, and cell apoptosis in HK-2 cells. Knockdown of orcl1 in podocytes reduced endocytosis and disrupted the cell cycle while increasing cell migration. In vivo studies in mice showed that conditional deletion of orcl1 in podocytes caused glomerular dysfunction, including proteinuria and fibrosis.
This study identified an R318H mutation in orcl1 in a patient with Dent-2 Disease. This mutation may contribute to renal injury by promoting ROS production and inducing cell apoptosis in tubular cells, while disrupting endocytosis and the cell cycle, and promoting cell migration of podocytes. Video Abstract.
摘要：
背景：本研究旨在鉴定患有Dent-2病的患者的orcl1突变，并研究其潜在机制。
方法：通过外显子组测序鉴定ocrl1突变。在HK-2和MPC5细胞中进行orcl1的敲低和orcl1突变体的过表达，以研究其功能。而流式细胞术测量活性氧（ROS），磷脂酰丝氨酸水平，和细胞凋亡。扫描电子显微镜观察晶体粘附，而透射电镜检查肾组织病理。使用激光扫描共聚焦显微镜检查内吞作用，免疫组织化学和免疫荧光法检测蛋白表达。此外,产生足细胞特异性orcl1基因敲除小鼠，以研究orcl1在体内的作用。
结果:我们鉴定了导致在先证者中ocrl1的位置318(R318H)处组氨酸被精氨酸取代的突变。orcl1在肾脏中广泛表达。体外实验表明，orcl1的敲低和ocrl1突变体的过表达增加了ROS，磷脂酰丝氨酸胞吐作用,晶体附着力，HK-2细胞凋亡。在足细胞中敲除orcl1减少内吞作用并破坏细胞周期，同时增加细胞迁移。小鼠体内研究表明足细胞中orcl1的条件性缺失导致肾小球功能障碍,包括蛋白尿和纤维化。
结论：本研究在患有Dent-2病的患者中鉴定了orcl1中的R318H突变。这种突变可能通过促进ROS产生和诱导肾小管细胞凋亡而导致肾损伤。同时破坏内吞作用和细胞周期，促进足细胞的细胞迁移。视频摘要。
方法：通过外显子组测序鉴定ocrl1突变。在HK-2和MPC5细胞中进行orcl1的敲低和orcl1突变体的过表达，以研究其功能。而流式细胞术测量活性氧（ROS），磷脂酰丝氨酸水平，和细胞凋亡。扫描电子显微镜观察晶体粘附，而透射电镜检查肾组织病理。使用激光扫描共聚焦显微镜检查内吞作用，免疫组织化学和免疫荧光法检测蛋白表达。此外,产生足细胞特异性orcl1基因敲除小鼠，以研究orcl1在体内的作用。
结果:我们鉴定了导致在先证者中ocrl1的位置318(R318H)处组氨酸被精氨酸取代的突变。orcl1在肾脏中广泛表达。体外实验表明，orcl1的敲低和ocrl1突变体的过表达增加了ROS，磷脂酰丝氨酸胞吐作用,晶体附着力，HK-2细胞凋亡。在足细胞中敲除orcl1减少内吞作用并破坏细胞周期，同时增加细胞迁移。小鼠体内研究表明足细胞中orcl1的条件性缺失导致肾小球功能障碍,包括蛋白尿和纤维化。
结论：本研究在患有Dent-2病的患者中鉴定了orcl1中的R318H突变。这种突变可能通过促进ROS产生和诱导肾小管细胞凋亡而导致肾损伤。同时破坏内吞作用和细胞周期，促进足细胞的细胞迁移。视频摘要。
