Fig 2: STAT1 mediates mTORC1-upregulated CFB expression in podocytes.(A and B) Representative immunohistochemical staining images showing the induction of p-STAT1 (Tyr701) and p-STAT1 (Ser727) in glomerulus from DKD human renal biopsies and mouse models. Scale bar: 20 µm. (C and D) Western blot assay showing the abundance of p-STAT1 (Tyr701) and p-STAT1 (Ser727) in mouse kidneys. (E) Representative immunohistochemical staining images showing the induction of p-STAT1 (Tyr701) and p-STAT1 (Ser727) in glomeruli among groups. Scale bar: 20 µm. (F) Western blot assay showing the abundance of p-S6, p-p70 S6K, p-4E-BP1, and p-mTOR in high glucose–cultured podocytes. (G) Western blot assay showing the abundance of p-STAT1 (Tyr701), p-STAT1 (Ser727), and CFB in high glucose–cultured podocytes treated with rapamycin (5 nM). (H) Representative immunofluorescence staining showing the abundance of p-S6, p-STAT1 (Ser727), and CFB in high glucose–cultured podocytes. Scale bar: 20 µm. (I and J) Western blot assay showing the abundance of CFB (I), p-S6, p-STAT1 (Tyr701), and p-STAT1 (Ser727) (J) in cultured podocytes treated with mTOR activator. (K) Western blot assay showing the abundance of p-STAT1 (Tyr701), p-STAT1 (Ser727), and CFB in cultured podocytes treated with fludarabine and mTOR activator (2 µM). (L) Western blot assay showing the abundance of p-STAT1 (Tyr701), p-STAT1 (Ser727), and CFB in high glucose–cultured podocytes treated with fludarabine (0.4 µM). (M) Quantitative analyses showing the abundance of CFB in high glucose–cultured podocytes treated with fludarabine (0.4 µM). #P < 0.05, *P < 0.05, n = 3. (N) Western blot assay showing the abundance of p-STAT1 (Tyr701), p-STAT1 (Ser727), and CFB in cultured podocytes treated with STAT1 siRNA and mTOR activator (2 µM). (O) Western blot assay showing the abundance of p-STAT1 (Tyr701), p-STAT1 (Ser727), and CFB in high glucose–cultured podocytes treated with STAT1 siRNA. Data are expressed as the mean ± SEM. Comparison between the groups was performed using 1-way ANOVA followed by the Tukey test.

Fig 3: Blocking mTORC1 with rapamycin attenuates alternative complement pathway activation and podocyte injury in STZ-induced diabetic mice.(A) The strategy for STZ injection and rapamycin administration. (B) UACR among different groups. n = 5. (C) Representative immunohistochemical staining images for p-S6, CFB, C3d, C5b-9, and C5aR in glomerulus among different groups. Representative PAS staining and immunofluorescence staining for diabetic kidney injury, WT1, and nephrin among different groups. Scale bar: 20 µm. (D–J) Quantitative analyses of p-S6, CFB, C3d, C5b-9, C5aR, and WT1-positive podocytes per glomerulus and glomerular area among different groups. *P < 0.05, n = 4–7. Data are expressed as the mean ± SEM. Comparison between the groups was performed using the 2-tailed Student’s t test (unpaired t test).

Fig 4: Activation of alternative complement pathway in glomeruli from DKD patients and mice.(A) Heatmap of the differentially expressed genes enriched in alternative complement pathway in glomeruli from human kidneys with DKD. (B) Representative transmission electron microscopy (TEM) pictures showing podocyte foot process effacement and glomerular basement membrane (GBM) thickness, represented by red text, in patients with DKD. Scale bars: 2 µm (above), 1 µm (below). (C) Representative immunohistochemical staining images showing the induction of CFB, C3d, C5b-9, and C5aR in glomerulus and tubulointerstitium from DKD renal biopsies. Scale bar: 20 µm. (D) Quantitative analyses of glomerular area in DKD renal biopsies. *P < 0.05 (control, n = 6; DKD, n = 34). (E) Quantitative analyses of CFB-staining-positive podocytes per glomerulus in renal biopsies. *P < 0.05 (control, n = 6; DKD, n = 34). (F) Linear correlation and regression analyses showing a significant positive correlation between glomerular area and CFB expression in DKD renal biopsies (n = 34). (G) The strategy for establishing a mouse model of DKD. (H) UACR 3 and 6 months after DM. *P < 0.05, n = 4. (I and J) UACR from db/db mice 3 and 6 months after DM. *P < 0.05, n = 3. (K) Representative immunohistochemical staining images showing the induction of CFB, C3d, C5b-9, and C5aR in glomeruli of STZ-treated mice and db/db mice. Scale bar: 20 µm. (L) Representative periodic acid–Schiff (PAS) staining and representative immunofluorescence staining revealing a decreased WT1-positive podocyte number and reduced nephrin abundance in STZ-treated mice and db/db mice. Scale bar: 20 µm. Data are expressed as the mean ± SEM. Comparison between the groups was performed using the 2-tailed Student’s t test (unpaired t test). Pearson’s correlation was used to determine relationships between variables. UACR, urinary albumin-to-creatinine ratio.

Fig 5: PP2Aca deficiency mediates high glucose–induced mTORC1 activation and CFB upregulation in podocytes.(A) Western blot assay showing the abundance of PP2Aca in cultured podocytes after high glucose treatment at different times. Relative change of PP2Aca/tubulin shown at the bottom of the bands. (B) Real-time PCR analysis showing the mRNA abundance for PP2Aca in cultured podocytes after high glucose treatment. *P < 0.05 and #P < 0.05 vs. control cells, n = 4. (C) Western blot assay showing the abundance of PP2Aca in cultured podocytes pretreated with lactacystin for 30 minutes, followed by high glucose treatment at different times. Relative change of PP2Aca/tubulin shown under the bands. (D) The cultured podocytes were pretreated with lactacystin for 30 minutes, followed by high glucose treatment for 15 minutes. Western blot assay showing the level of ubiquitin in the precipitates (PP2Aca-IP) and PP2Aca in lysates (input). (E) Western blotting assay showing the expression of PP2Aca (MR204384) in cultured podocytes after PP2Aca (MR204384) plasmid transfection. (F and G) Western blotting assays showing the abundance of p-S6, p-p70 S6K, p-4E-BP1, p-mTOR (F), p-Stat1 (Ser727), and CFB (G) in high glucose–cultured podocytes transfected with pcDNA3.0 or PP2Aca (MR204384) plasmid for 36 hours. (H) Western blotting analyses demonstrating the downregulation of PP2Aca after PP2Aca siRNA transfection. (I and J) Western blotting assays showing the abundance of p-S6, p-p70 S6K, p-4E-BP1, p-mTOR (I), p-Stat1 (Ser727), and CFB (J) in high glucose–cultured podocytes transfected with scramble or PP2Aca siRNA for 36 hours. Data are expressed as the mean ± SEM. Comparison between the groups was performed using 1-way ANOVA followed by the Tukey test.