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. 2023 Jul;46(7):1759-1770.
doi: 10.1038/s41440-023-01300-3. Epub 2023 May 15.

Endothelial-derived complement factor D contributes to endothelial dysfunction in malignant nephrosclerosis via local complement activation

Zheng Wang  1   2 , Zhe Zhang  1   2 , Yuan Li  1 , Ying Zhang  1 , Min Wei  1 , Hui Li  1   2 , Shanzhi Yang  1 , Yali Zhou  1 , Xinjin Zhou  3   4 , Guolan Xing  5
Affiliations

Endothelial-derived complement factor D contributes to endothelial dysfunction in malignant nephrosclerosis via local complement activation

Zheng Wang et al. Hypertens Res. 2023 Jul.

Abstract

Malignant nephrosclerosis is a thrombotic microangiopathy associated with abnormal local activation of the complement alternative pathway (AP). However, the mechanism underlying local AP activation is not fully understood. We hypothesized that complement factor D (CFD) secreted by endothelial cells triggers vascular dysfunction in malignant nephrosclerosis via local complement activation. We investigated the deposition of CFD in human kidney biopsy tissues and the function of endothelial-derived CFD in endothelial cell cultures. Immunofluorescence microscopy and laser microdissection-targeted mass spectrometry revealed significant deposition of CFD in the kidneys of patients with malignant nephrosclerosis. Conditionally immortalized human glomerular endothelial cells (CiGEnCs) continuously expressed and secreted CFD in vitro. CFD knockdown in CiGEnCs by small interfering RNA reduced local complement activation and attenuated the upregulation of intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), von Willebrand factor (VWF), and endothelin-1 (ET-1) induced by Ang II. The expression of CFD in CiGEnCs was significantly higher than that in other types of microvascular endothelial cells. Our findings suggest that (i) glomerular endothelial cells are an important source of local renal CFD, (ii) endothelial-derived CFD can activate the local complement system, and (iii) endothelial-derived CFD mediates endothelial dysfunction, which may play a role in the pathogenesis of malignant nephrosclerosis.

Keywords: Alternative pathway; Complement factor D; Endothelial cells; Local complement; Malignant nephrosclerosis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Immunofluorescence and targeted mass spectrometry quantification of renal specimens from patients with malignant nephrosclerosis. A IF microscopy showing the localization of CFD (red), CD34 (green), and DAPI (blue) and merged image showing the co-localization of CFD and CD34 (yellow). B IF microscopy showing the localization of C5b-9 (red), CD34 (green), and DAPI (blue) and merged image showing the co-localization of C5b-9 and CD34 (yellow). C IF microscopy showing the localization and Intensity of CFB, C3 and C4. D Area under the curve (AUC) of RPDSLQHVLLPVLDR, ATLGPAVRPLPWQR, and VQVLLGAHSLSQPEPSK (unique peptides of CFD) showing the relative abundance and spectra showing the co-eluted fragment ions of the corresponding peptides. E AUC of ALPTTYEK, LSPIYNLVPVK, LYYGDDEK, and LPLEYSYGEYR (unique peptides of C5b-9) (C8a, C8b, and C9) showing the relative abundance and spectra showing the co-eluted fragment ions of the corresponding peptides. All data are the mean ± SD; n ≥ 3 per group; *p < 0.05 compared with the expression in the negative control group. Scale bars represent 50 μm
Fig. 2
Fig. 2
Cell viability of CiGEnCs and expression of ICAM-1, VCAM-1, vWF, and ET-1 in CiGEnCs stimulated with different concentrations of Ang II. A CCK-8 analysis of the cell viability of endothelial cells stimulated under different conditions. B mRNA expression of ICAM-1, VCAM-1, vWF, and ET-1 in endothelial cells incubated for 24 h with the negative control (white bars), 0.1 μm Ang II (gray bars), and 1 μm Ang II (black bars). C, D Protein expression of ICAM-1, VCAM-1, vWF, and ET-1 in cultured endothelial cells after stimulation with different concentrations of Ang II. All data are the mean ± SD; n ≥ 3 per group; *p < 0.05, **p < 0.01, ***p < 0.001 compared with the expression in the negative control group
Fig. 3
Fig. 3
Expression and secretion of CFD by CiGEnCs. A, D Protein expression of CFD in CiGEnCs after stimulation with different concentrations of Ang II. B Secretion of CFD in the supernatant of CiGEnCs stimulated with different concentrations of Ang II for 12 h, 24 h, and 36 h. C mRNA expression of CFD in CiGEnCs incubated for 24 h with the negative control (white bars), 0.1 μm Ang II, (gray bars), and 1 μm Ang II (black bars). E Quantitative analysis of western blots for the secretion of CFD from CiGEnCs incubated in serum-free medium, 0.1 μM Ang II, and 1 μM Ang II for 0 h (white bars), 12 h (light gray bars), 24 h (dark gray bars), and 36 h (black bars). All data are the mean ± SD; n ≥ 3 per group; **p < 0.01 and ***p < 0.001 compared with secretion with serum-free medium; #p < 0.05 and ###p < 0.001 compared with incubation with 0.1 μM Ang II
Fig. 4
Fig. 4
Reduction in local complement activation with gene silencing of CFD in CiGEnCs in vitro. A mRNA expression of CFD in CiGEnCs cultured for 24 h. B IF diagram showing the localization of C5b-9 (green), CD34 (red) and DAPI (blue). The merged image showing the colocalization of C5b-9 and CD34 (yellow). C Secretion of CFD in the supernatant of CiGEnCs cultured for 24 h. D Quantitative analysis of western blots for the secretion of CFD from CiGEnCs cultured for 24 h. SFM serum-free medium; All data are the mean ± SD; n ≥ 3 per group; ***p < 0.001 compared with the expression in the mock group. Scale bars represent 20 μm
Fig. 5
Fig. 5
Attenuation of the Ang II-induced upregulation of ICAM-1, VCAM-1, vWF, and ET-1 expression in CiGEnCs with CFD knockdown. A, D, G, J Effects of CFD knockdown on the mRNA expression of ICAM-1, VCAM-1, vWF, and ET-1 in CiGEnCs. B, E, H, K Representative western blots showing changes in the protein levels of ICAM-1, VCAM-1, vWF, and ET-1 in CiGEnCs after CFD knockdown. C, F, I, L Protein levels of ICAM-1, VCAM-1, vWF, and ET-1 in CiGEnCs of each group shown as a ratio relative to β-actin. All data are the mean ± SD; n ≥ 3 per group; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the mock group; #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the group transfected with siRNA-negative control and stimulated with Ang II
Fig. 6
Fig. 6
Differential expression of CFD in CiGEnCs and HMECs. A mRNA expression levels of complement AP components in conventionally cultured CiGEnCs and HMECs. B mRNA expression levels of complement AP components in CiGEnCs and HMECs stimulated with Ang II. C, D Protein expression levels of CFD in conventionally cultured CiGEnCs and HMECs. All data are the mean ± SD; n ≥ 3 per group; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with HMECs
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