#2382 Renal tubule-derived EVs carrying complement 3 aggravate CKD vascular calcification by downregulating autophagy in vascular smooth muscle cells

Abstract

Abstract Background and Aims Vascular calcification (VC) is a serious complication of chronic kidney disease (CKD) patients, recognized in KDIGO guidelines as a leading risk factor for cardiovascular diseases. Despite acknowledgment, the mechanisms of VC in CKD remain unclear. The kidneys intricately regulate blood vessel pathophysiology. Clinical studies suggest a correlation between elevated blood complement levels in CKD and arterial calcification, hinting at complement involvement in CKD-associated VC. Renal-origin complement, a significant source of circulating complement, activates in CKD. Extracellular vesicles (EVs), mediating intercellular communication, transport complement-related proteins in various physiological and pathological processes. Studies suggest that plasma EV rather than EV-depleted plasma causes pathogenic calcium salt deposition in smooth muscle cells. This study explores how renal-origin EVs carrying complement mediate CKD-associated VC. Method From October 2021 to August 2023, data from 110 clinical patients were gathered, encompassing both blood C3 levels and CT images of the thoracic aorta. Subsequently, we conducted a scoring analysis of the CT images and carried out correlation analyses. A CKD-associated VC model was induced by feeding C57BL/6j mice an adenine and high-phosphate diet for 16 weeks. Techniques, including WB, PCR, and immunofluorescence, detected complement activation and localization in renal tissues. EVs were isolated from kidney tissue and plasma and identified by NTA, electron microscopy, WB, etc. WB examined C3 and tubular markers of EVs. At the same time, the Elisa method was used to detect C3 in plasma, plasma EV, and EV-depleted plasma. Immunofluorescence investigated tubular-derived EVs uptake in calcified arteries. Tubular-derived EVs, C3a, C3aR receptor inhibitors (SB290157), and autophagy inhibitors (3-MA) intervened in high-phosphate-induced vascular smooth muscle cells (VSMCs). PCR and WB assessed calcification indicators, complement receptors, and autophagy-related indicators. Alizarin Red staining observed calcification nodules. Transcriptome sequencing was performed on control, high-phosphate, and renal tubular-derived EV intervention groups of VSMCs. An in vivo model infused renal tubular-derived EVs and injected C3aR inhibitors and autophagy inhibitors, measuring complement activation and VC indicators. Results There is a correlation between C3 and thoracic aorta calcium score in CKD patients, with a correlation coefficient of 0.3750 (P < 0.01). The CKD-associated VC mice model revealed complement C3 activation in renal tubules, secreted into the blood through EVs. The C3 proportion of plasma EV in CKD patients was significantly higher than that in the control group, but there was no statistical difference between the EV-depleted plasma control group and the CKD group. Immunofluorescence indicated tubular-derived EV uptake by the calcified vascular wall. In vitro, tubular-derived EVs enhanced VSMC osteogenic differentiation and upregulated C3aR, mimicking C3a intervention effects. C3aR receptor inhibitors produced opposing effects. Transcriptome sequencing suggested renal tubular-derived EVs downregulated VSMC autophagy-related pathways; in vitro autophagy inhibition reversed osteogenic differentiation. In vivo, reinfusing renal tubular-derived EVs exacerbated complement activation and VC, while injecting C3aR inhibitors and autophagy inhibitors alleviated CKD-associated VC. Conclusion This study confirms that tubular-derived EVs carrying C3 downregulate autophagy in VSMCs, mediating CKD-associated VC. These findings provide insights into understanding CKD-associated VC initiation and progression, revealing potential therapeutic targets.