TangShenWeiNing Formula Prevents Diabetic Nephropathy by Protecting Podocytes Through the SIRT1/HIF-1α Pathway

Abstract

Background: Diabetic nephropathy (DN) represents a major complication of diabetes, and podocyte injury has a critical function in DN development. TangShenWeiNing formula (TSWN) has been demonstrated to efficiently decrease proteinuria and protect podocytes in DN. This work aimed to explore the mechanism by which TSWN alleviates DN and protects podocytes.

Methods: The major bioactive components of TSWN were detected by mass spectrometry (MS) and pharmacological databases. Eight-week-old male C57BLKS/J db/m and db/db mice were provided pure water, valsartan, low dose TSWN, middle dose TSWN and high dose TSWN by gavage for 12 weeks, respectively.

Results: MS and network pharmacology analyses suggested that TSWN might prevent DN through the sirtuin (SIRT)1/hypoxia-inducible factor (HIF)-1α pathway. Diabetic mice showed elevated urinary albumin in comparison with non-diabetic mice, and TSWN decreased urinary albumin in diabetic mice. Histological injury increased in the kidney in diabetic mice, which could be improved by TSWN. Fibrosis and collagen I expression were induced in the diabetic mouse kidney in comparison with the non-diabetic mouse kidney; TSWN alleviated these effects. Apoptosis and cleaved caspase-3 were induced in the diabetic mouse kidney in comparison with the non-diabetic mouse kidney, and TSWN blunted these effects. Podocytes were damaged in the diabetic mouse kidney, which was improved by TSWN. Podocin and nephrin amounts were decreased in the diabetic mouse kidney in comparison with the non-diabetic mouse kidney, and podocalyxin was increased in urine of diabetic animals in comparison with non-diabetic counterparts. After TSWN treatment, podocin and nephrin were raised in the diabetic mouse kidney, and urinary podocalyxin was depressed in diabetic animals. Diabetic mice had lower SIRT1 and higher HIF-1α amounts in kidney specimens in comparison with non-diabetic mice, and TSWN promoted SIRT1 and inhibited HIF-1α in the diabetic mouse kidney. Moreover, co-staining of SIRT1 and podocin revealed that SIRT1 decreased in podocytes from diabetic mice in comparison with those from non-diabetic mice, and TSWN elevated SIRT1 in podocytes.

Conclusions: This study indicated that TSWN alleviates DN by improving podocyte injury through the SIRT1/HIF-1α pathway in diabetic mouse kidneys.

Keywords: HIF-1α; SIRT1; TangShenWeiNing formula; diabetic nephropathy; podocytes (MeSH: D050199).

Figure 1

Network pharmacological analyses. (A) Venn diagram of targets of TSWN formula and targets of diabetic nephropathy. (B) Pathway enrichment analysis of differential genes by TSWN formula treatment on diabetic nephropathy. The vertical axis represents the names of the 20 selected pathways, the color of the dot represents the -log10(P) value, the size represents the number of genes, and the horizontal axis is the enrichment of pathways.

Figure 2

Physical and biochemical characteristics of mice. (A) Body weight. (B) Kidney weight. (C) Food intake. (D) Fasting blood glucose level. (E) Fasting Insulin level. (F) Fasting total cholesterol. (G) Fasting triglycerides. (H) Alanine aminotransferase (ALT). (I) Blood urea nitrogen (BUN). (J) Serum creatinine (SCr). n = 6 mice/group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 vs db/m group. Db/m, db/m mice; db/db, db/db mice; db/db+V, db/db mice with valsartan treatment; db/db+TSWN-L, db/db mice with low dose TSWN treatment; db/db+TSWN-M, db/db mice with middle dose TSWN treatment; db/db+TSWN-H, db/db mice with high dose TSWN treatment. Data are means ± S.E.M.

Figure 3

Renal phenotype of mice. (A) Urinary albumin excretion (UAE). (B) Urinary albumin-to-creatinine ratio (UACR). (C) Representative photographs of mouse kidneys by Hematoxylin & Eosin (H&E) staining. (D) Representative photographs of mouse kidneys by Periodic Acid Schiff (PAS) staining. n = 6 mice/group. ^####P < 0.0001 vs db/m group; **P < 0.01 vs db/db group. Db/m, db/m mice; db/db, db/db mice; db/db+V, db/db mice with valsartan treatment; db/db+TSWN-L, db/db mice with low dose TSWN treatment; db/db+TSWN-M, db/db mice with middle dose TSWN treatment; db/db+TSWN-H, db/db mice with high dose TSWN treatment. Data are means ± S.E.M.

Figure 4

Fibrosis in mouse kidneys. (A) Representative photographs of renal fibrosis measured by Masson’s staining. (B) Representative photographs of renal fibrosis measured by Sirius red staining. (C) Quantification of renal fibrosis. (D) Representative photographs and quantification of collagen I in mouse kidneys detected by western blot. n = 6 mice/group. ^###P < 0.001, ^####P < 0.0001 vs db/mgroup; *P < 0.05, **P < 0.01, ****P < 0.0001 vs db/db group. Db/m, db/m mice; db/db, db/db mice; db/db+V, db/db mice with valsartan treatment; db/db+TSWN-L, db/db mice with low dose TSWN treatment; db/db +TSWN-M, db/db mice with middle dose TSWN treatment; db/db+TSWN-H, db/db mice with high dose TSWN treatment. Data are means ± S.E.M.

Figure 5

Apoptosis in mouse kidneys. (A, B) Representative photographs and quantification of renal apoptosis measured by TUNEL assay. (C) Representative photographs and quantification of cleaved caspase-3 in mouse kidneys detected by western blot. n = 6 mice/group. ^####P < 0.0001 vs db/m group; **P < 0.01, ***P < 0.001, ****P < 0.0001 vs db/db group. Db/m, db/m mice; db/db, db/db mice; db/db+V, db/db mice with valsartan treatment; db/db+TSWN-L, db/db mice with low dose TSWN treatment; db/db+TSWN-M, db/db mice with middle dose TSWN treatment; db/db+TSWN-H, db/db mice with high dose TSWN treatment. Data are means ± S.E.M.

Figure 6

Podocytes in mouse kidneys. (A) Representative photographs and quantification of podocin in mouse kidneys measured by western blot. (B) Representative photographs and quantification of nephrin in mouse kidneys measured by western blot. (C) Quantification of urinary podocalyxin measured by enzyme linked immunosorbent assay (ELISA). (D) Representative photographs of podocytes in mouse kidneys detected by transmission electron microscopy. n = 6 mice/group. ^####P < 0.0001 vs db/m group; *P < 0.05, **P < 0.01, ****P < 0.0001 vs db/db group. Db/m, db/m mice; db/db, db/db mice; db/db+V, db/db mice with valsartan treatment; db/db+TSWN-L, db/db mice with low dose TSWN treatment; db/db+TSWN-M, db/db mice with middle dose TSWN treatment; db/db+TSWN-H, db/db mice with high dose TSWN treatment. Data are means ± S.E.M.

Figure 7

SIRT1 and HIF-1a in mouse kidneys. (A) Messenger ribonucleic acid (mRNA) expression of SIRT1 in mouse kidneys by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). (B) MRNA expression of HIF-1a in mouse kidneys by RT-PCR. (C) Representative photographs and quantification of SIRT1 in mouse kidneys measured by western blot. (D) Representative photographs and quantification of HIF-1a in mouse kidneys measured by western blot. (E–G) Representative photographs and quantification of co-staining of SIRT1 and podocin in mouse kidneys. n = 6 mice/group. ^####P < 0.0001 vs db/m group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs db/db group. Db/m, db/m mice; db/db, db/db mice; db/db+V, db/db mice with valsartan treatment; db/db+TSWN-L, db/db mice with low dose TSWN treatment; db/db+TSWN-M, db/db mice with middle dose TSWN treatment; db/db+TSWN-H, db/db mice with high dose TSWN treatment. Data are means ± S.E.M.