Tangshen Formula Attenuates Diabetic Nephropathy by Promoting ABCA1-Mediated Renal Cholesterol Efflux in db/db Mice

Abstract

The commonly prescribed Tangshen Formula (TSF) is a traditional Chinese formulation that has been shown to reduce plasma lipid metabolism and proteinuria and improve the estimated glomerular filtration rate (eGFR) in patients with diabetic kidney disease. This study investigated the underlying mechanism whereby TSF regulates renal lipid accumulation and ameliorates diabetic renal injuries in spontaneous diabetic db/db mice and in vitro in sodium palmitate (PA)-stimulated and Abca1-SiRNA-transfected mouse tubular epithelial cells (mTECs). The results revealed that TSF treatment significantly ameliorated the renal injuries by lowering urinary albumin excretion and improving renal tissue injuries in diabetic (db/db) mice. Interestingly, the treatment with TSF also resulted in decreased cholesterol levels in the renal tissues of db/db mice, which was associated with increased expression of the peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), the Liver X receptors (LXR), and ATP-binding cassette subfamily A member 1 (ABCA1), suggesting that TSF might attenuate diabetic kidney injury via a mechanism associated with improving cholesterol efflux in the diabetic kidney. This was investigated in vitro in mTECs, and the results showed that TSF reduced the PA-stimulated cholesterol accumulation in mTECs. Mechanistically, the addition of TSF was capable of reversing PA-induced downregulation of PGC-1α, LXR, and ABCA1 expression and cholesterol accumulation in mTECs, suggesting that TSF might act the protection via the PGC-1α-LXR-ABCA1 pathway to improve the cholesterol efflux in the renal tissues of db/db mice. This was further confirmed by silencing ABCA1 to block the promotive effect of TSF on cholesterol efflux in vitro. In conclusion, TSF might ameliorate diabetic kidney injuries by promoting ABCA1-mediated renal cholesterol efflux.

Keywords: ABCA1; Abca1-SiRNA; Tangshen formula (TSF); diabetic nephropathy (DN); renal cholesterol efflux.

Figure 1

TSF ameliorated renal injuries in db/db mice. Administration of TSF effectively reduced body weight (A) and UACR (B). (C) TSF reduced dyslipidemia in 22-week-old db/db mice. The data were expressed as the mean ± SEM. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. the db/m group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the db/db group.

Figure 2

TSF attenuated glomerular mesangial matrix deposition, and lipid and cholesterol accumulation in the renal tissues of db/db mice. (A) PAS staining (bar = 25 μm). (B) Oil Red O staining (bar = 50 μm). (C) Filipin cholesterol staining (bar = 25 μm). (D) Analysis with a colorimetric assay demonstrated that TSF decreased total cholesterol levels in the renal tissues of 22-week-old db/db mice. ^**P < 0.01, ^***P < 0.001 vs. the db/m group; ^#P < 0.05 vs. the db/db group.

Figure 3

After treatment with TSF, the expression of ABCA1, PGC-1α, and LXR in renal tissues was upregulated in db/db mice. (A) Effect of TSF on protein levels of ABCA1, PGC-1α, and LXR by Western blot analysis. (B) Effect of TSF on mRNA expression of ABCA1, PGC-1α, and LXR real-time PCR analysis. The data were represented as the mean ± SEM for at least three independent experiments. ^*P < 0.05 and ^***P < 0.001 vs. the db/m group; ^#P < 0.05, ^###P < 0.001 vs. the db/db group.

Figure 4

TSF prevented cholesterol accumulation in the mTECs stimulated with PA. (A) Dose-dependent effect of TSF on cell viability as determined by the MTT assay for 24 and 48 h. ^**P < 0.01 and ^***P < 0.001 vs. the medium group. (B) A colorimetric analysis demonstrated that TSF decreased total cholesterol levels in the mTECs stimulated with PA for 24 and 48 h. ^**P < 0.01 and ^***P < 0.001 vs. the PA group. The data were represented as the mean ± SEM for at least three independent experiments.

Figure 5

After treatment with TSF, the expression of PGC-1α, LXR, and ABCA1 was upregulated in the mTECs stimulated with PA. (A) Western blot analysis of PGC-1α and LXR expression in mTECs cultured for 12 h. (B) Western blot analysis of ABCA1 expression in mTECs cultured for 48 h. (C) Real-time PCR analysis of PGC-1α and LXR expression in mTEC cells cultured for 3 h. (D) Real-time PCR analysis of ABCA1 expression in mTEC cells cultured for 6 h. The data were represented as the mean ± SEM for at least three independent experiments. ^*P < 0.05, ^**P < 0.01 vs. the PA group.

Figure 6

Silencing ABCA1 significantly suppressed the inhibitory effect of TSF on PA-induced total cholesterol levels in mTECs. (A) Western blot analysis of ABCA1, PGC-1α, and LXR expression in mTECs cultured for 48 h, and then exposed to OA (oleic acid 50 μM), PA (sodium palmitate 50 μM), PA+T (PA 50 μM+ TSF 500 μg/mL), PA+T+A (PA 50 μM+ TSF 500 μg/mL+ Abca1-SiRNA), or PA+T+A (PA 50 μM+ TSF 500 μg/mL+ Co-Abca1-SiRNA). (B) Real-time PCR analysis of ABCA1, PGC-1α, and LXR expression in mTECs cultured for 6 h. (C) A colorimetric analysis of total cholesterol levels in the mTECs cultured with Abca1-SiRNA for 24 and 48 h. ^*P < 0.05, ^**P < 0.01 vs. the PA group; ^#P < 0.05, ^##P < 0.01 vs. the PA+T+A group.