Qufeng tongluo decoction decreased proteinuria in diabetic mice by protecting podocytes via promoting autophagy

Abstract

Background: Diabetic kidney disease (DKD) is one of diabetic complications, which has become the leading cause of end-stage kidney disease. In addition to angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker(ACEI/ARB) and sodium-glucose cotransporter-2 inhibitor (SGLT2i), traditional Chinese medicine (TCM) is an effective alternative treatment for DKD. In this study, the effect of Qufeng Tongluo (QFTL) decoction in decreasing proteinuria has been observed and its mechanism has been explored based on autophagy regulation in podocyte.

Methods: In vivo study, db/db mice were used as diabetes model and db/m mice as blank control. Db/db mice were treated with QFTL decoction, rapamycin, QFTL + 3-Methyladenine (3-MA), trehalose, chloroquine (CQ) and QFTL + CQ. Mice urinary albumin/creatinine (UACR), nephrin and autophagy related proteins (LC3 and p62) in kidney tissue were detected after intervention of 9 weeks. Transcriptomics was operated with the kidney tissue from model group and QFTL group. In vitro study, mouse podocyte clone-5 (MPC-5) cells were stimulated with hyperglycemic media (30 mmol/L glucose) or cultured with normal media. High-glucose-stimulated MPC-5 cells were treated with QFTL freeze-drying powder, rapamycin, CQ, trehalose, QFTL+3-MA and QFTL + CQ. Cytoskeletal actin, nephrin, ATG-5, ATG-7, Beclin-1, cathepsin L and cathepsin B were assessed. mRFP-GFP-LC3 was established by stubRFP-sensGFP-LC3 lentivirus transfection.

Results: QFTL decoction decreased the UACR and increased the nephrin level in kidney tissue and high-glucose-stimulated podocytes. Autophagy inhibitors, including 3-MA and chloroquine blocked the effects of QFTL decoction. Further study showed that QFTL decoction increased the LC3 expression and relieved p62 accumulation in podocytes of db/db mice. In high-glucose-stimulated MPC-5 cells, QFTL decoction rescued the inhibited LC3 and promoted the expression of ATG-5, ATG-7, and Beclin-1, while had no effect on the activity of cathepsin L and cathepsin B. Results of transcriptomics also showed that 51 autophagy related genes were regulated by QFTL decoction, including the genes of ATG10, SCOC, ATG4C, AMPK catalytic subunit, PI3K catalytic subunit, ATG3 and DRAM2.

Conclusion: QFTL decoction decreased proteinuria and protected podocytes in db/db mice by regulating autophagy.

Keywords: Autophagy; Podocyte; Proteinuria; Qufeng tongluo decoction; Traditional Chinese medicine.

Graphical abstract

Fig. 1

QFTL decoction relieved proteinuria and protected the podocyte. (a)UACR (n = 6–9) of db/db mice was relieved by QFTL decoction and autophagy activator rapamycin. Autophagy inhibitors including 3-MA and chloroquine blunted the effect of QFTL decoction. (b)IHC results (n = 4) showed that db/db mice had less nephrin compared with db/m mice, and QFTL decoction and rapamycin rescued the inhibited nephrin expression. 3-MA and chloroquine blunted the effect of QFTL decoction. (c-d) Similar WB results of nephrin were observed in kidney tissue (n = 6) and MPC-5 cell lines (n = 3). (e)Phalloidin staining results showed that QFTL decoction could protect the cytoskeleton of high-glucose-simulated MCP-5 cells as well as rapamycin, and autophagy inhibitors blunted the effect of QFTL decoction. UACR, urinary albumin creatinine ratio; 3-MA, 3-Methyladenine; CQ, chloroquine. *, p ≤ 0.05.

Fig. 2

StubRFP-sensGFP-LC3 lentivirus transfection in high-glucose-stimulated podocytes. Yellow puncta decreased after high glucose stimulation and rescued by QFTL decoction as well as rapamycin, but not trehalose. Puncta decreased when co-intervened with 3-MA compared with those treated with QFTL decoction alone.

Fig. 3

LC3 and p62 expression in podocytes of db/db mice. (a) Immunofluorescence staining (n = 6–8) showed that fewer LC3 puncta were observed in db/db mice and QFTL decoction could increase LC3 expression. 3-MA blunted the effect of QFTL decoction. (b) p62 accumulation (n = 6–8) in podocytes was observed in db/db mice and was relieved by QFTL decoction. Chloroquine blunted the effect of QFTL decoction in relieving p62 accumulation. (c) Western blot results (n = 6) showed a similar trend of LC3 and p62, and chloroquine couldn't regulate them as QFTL did. *, p ≤ 0.05.

Fig. 4

QFTL decoction promoted the formation of autophagosomes in high-glucose-stimulated podocytes. (a) Immunofluorescence staining images and WB results (n = 3) showed that ATG-5 was inhibited by high-glucose intervention and could be rescued by QFTL decoction. Chloroquine didn't increase ATG-5 as QFTL did. (b-c) Similar results (n = 3) were observed for ATG-7 and beclin-1. Chloroquine didn't increase ATG-7 and Beclin-1 as QFTL did. (d) The activity of cathepsin L and cathepsin B in MCP-5 was detected by ELISA kit. Results (n = 3) showed that QFTL decoction had no significant effect on the activity of cathepsin L and cathepsin B. (e) Results of transcriptomics (n = 4) showed that genes of SCOC, ATG-10, ATG-4C, ATG-3, DRAM-2, VPS-41, PK3CD, and AAPK2 were regulated by QFTL decoction. L, low-glucose; H, high-glucose; CB, cathepsin B; CL, cathepsin L. *, p ≤ 0.05.