Complement factor B in high glucose–induced podocyte injury and diabetic kidney disease
Qingmiao Lu, Qing Hou, Kai Cao, Xiaoli Sun, Yan Liang, Mengru Gu, Xian Xue, Allan Zijian Zhao, Chunsun Dai
Qingmiao Lu, Qing Hou, Kai Cao, Xiaoli Sun, Yan Liang, Mengru Gu, Xian Xue, Allan Zijian Zhao, Chunsun Dai
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Research Article Inflammation Nephrology

Complement factor B in high glucose–induced podocyte injury and diabetic kidney disease

Abstract

The role and mechanisms for upregulating complement factor B (CFB) expression in podocyte dysfunction in diabetic kidney disease (DKD) are not fully understood. Here, analyzing Gene Expression Omnibus GSE30528 data, we identified genes enriched in mTORC1 signaling, CFB, and complement alternative pathways in podocytes from patients with DKD. In mouse models, podocyte mTOR complex 1 (mTORC1) signaling activation was induced, while blockade of mTORC1 signaling reduced CFB upregulation, alternative complement pathway activation, and podocyte injury in the glomeruli. Knocking down CFB remarkably alleviated alternative complement pathway activation and DKD in diabetic mice. In cultured podocytes, high glucose treatment activated mTORC1 signaling, stimulated STAT1 phosphorylation, and upregulated CFB expression, while blockade of mTORC1 or STAT1 signaling abolished high glucose–upregulated CFB expression. Additionally, high glucose levels downregulated protein phosphatase 2Acα (PP2Acα) expression, while PP2Acα deficiency enhanced high glucose–induced mTORC1/STAT1 activation, CFB induction, and podocyte injury. Taken together, these findings uncover a mechanism by which CFB mediates podocyte injury in DKD.

Authors

Qingmiao Lu, Qing Hou, Kai Cao, Xiaoli Sun, Yan Liang, Mengru Gu, Xian Xue, Allan Zijian Zhao, Chunsun Dai

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Figure 6

Rheb1 is dispensable for high glucose–induced mTORC1 activation, and ablation of Rheb1 aggravates podocyte injury in streptozotocin-induced diabetic mice.

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Rheb1 is dispensable for high glucose–induced mTORC1 activation, and abl...
(A) Western blot assay showing the abundance of Rheb1 in cultured podocytes after high glucose treatment at different times. (B) GTP loading assay showing the induction of GTP-Rheb1 after high glucose treatment. (C) Western blot assay showing the abundance of p-S6, p-p70 S6K, p-4E-BP1, and p-mTOR in high glucose–cultured podocytes transfected with scramble or Rheb1 siRNA for 24 hours. (D and E) The cultured podocytes were pretransfected with scramble or Rheb1 siRNA for 24 hours, or treated with rapamycin for 30 minutes, followed by high glucose treatment for 24 hours. Propidium iodide (PI) staining (D) and quantitative analysis (E) of dead cells among different groups. Data are presented as the percentage of PI-staining-positive cells. #P < 0.05 vs. scramble control cells, n = 3; *P < 0.05, $P < 0.05 vs. high glucose–treated podocytes, n = 3. Scale bar: 20 μm. (F) UACR in Podo-Rheb1+/+ and Podo-Rheb1–/– mice at 6 months after STZ-induced DM. *P < 0.05 vs. Podo-Rheb1+/+ mice, n = 6–7. (G) Representative PAS staining and immunofluorescence staining for diabetic kidney injury, WT1, and nephrin among different groups. Scale bar: 50 μm. (H–J) Quantitative analyses of injury score, glomerular area, and WT1-positive podocytes per glomerulus among different groups. #P < 0.05 vs. vehicle control mice; *P < 0.05 vs. Podo-Rheb1+/+ mice with STZ injection, n = 3–7. Data are expressed as the mean ± SEM. Comparison between the groups was performed using 1-way ANOVA followed by the Tukey test (B and E). Comparison between the groups was performed using the 2-tailed Student’s t test (paired t test) (F). Data in H–J were analyzed with 2-way ANOVA with Tukey’s post hoc test.