Metformin ameliorates the severity of experimental Alport syndrome

Abstract

Metformin is widely used for the treatment of type 2 diabetes, and increasing numbers of studies have shown that metformin also ameliorates tumor progression, inflammatory disease, and fibrosis. However, the ability of metformin to improve non-diabetic glomerular disease and chronic kidney disease (CKD) has not been explored. To investigate the effect of metformin on non-diabetic glomerular disease, we used a mouse model of Alport syndrome (Col4a5 G5X) which were treated with metformin or losartan, used as a control treatment. We also investigated the effect of metformin on adriamycin-induced glomerulosclerosis model. Pathological and biochemical analysis showed that metformin or losartan suppressed proteinuria, renal inflammation, fibrosis, and glomerular injury and extended the lifespan in Alport syndrome mice. Transcriptome analysis showed that metformin and losartan influenced molecular pathways-related to metabolism and inflammation. Metformin altered multiple genes including metabolic genes not affected by losartan. Metformin also suppressed proteinuria and glomerular injury in the adriamycin-induced glomerulosclerosis mouse model. Our results showed that metformin ameliorates the glomerular sclerosis and CKD phenotype in non-diabetic chronic glomerular diseases. Metformin may have therapeutic potential for not only diabetic nephropathy but also non-diabetic glomerular disease including Alport syndrome.

Figure 1

Metformin protects against progressive renal dysfunction in Col4a5 G5X Alport syndrome mice. (a) The experimental design of studies performed on C57BL/6 Col4a5 G5X Alport syndrome mice is shown. The image was drawn by S.K. (b) Proteinuria score was calculated based on urinary protein and creatinine concentrations. Proteinuria was reduced in losartan or metformin-treated C57BL/6 Col4a5 G5X Alport syndrome mice. (c) Creatinine-normalized urinary albumin concentration was reduced in losartan- or metformin-treated C57BL/6 Col4a5 G5X Alport syndrome mice. (d) The elevation of serum creatinine level in late stage C57BL/6 Col4a5 G5X Alport syndrome mice was suppressed by losartan or metformin treatment. Data are expressed as the means ± S.E. in WT (n = 5), vehicle-, losartan- or metformin-treated C57BL/6 Col4a5 G5X Alport syndrome mice (n = 8–9 per group). P values were assessed by Dunnett’s test (^##P < 0.01 vs WT. *P < 0.05, **P < 0.01 vs vehicle).

Figure 2

Metformin suppresses renal inflammation and fibrosis in Col4a5 G5X Alport syndrome mice. (a) Staining of renal sections of 20-week-old mice by PAS, Masson-Trichrome, and F4/80 immunohistochemistry (IHC) indicated renal inflammation was ameliorated in losartan- or metformin-treated C57BL/6 Col4a5 G5X Alport syndrome mice. Scale bars, PAS 50 μm; MT and F4/80 200 μm. (b) Glomerular injury scores were evaluated based on the PAS-stained sections. The severity of glomerulosclerosis was decreased in losartan- or metformin-treated C57BL/6 Col4a5 G5X Alport syndrome mice. (c) Tubulointerstitial fibrosis scores were evaluated based on the MT-stained sections. The fibrotic region was reduced in losartan- or metformin-treated C57BL/6 Col4a5 G5X Alport syndrome mice. (d) F4/80-positive region was evaluated based on the F4/80 IHC section. Both losartan and metformin suppressed the infiltration of macrophages. (e–l) Total RNA was isolated from renal tissues of 20-week-old mice, and subjected to quantitative RT-PCR. The data were normalized to Gapdh. Data are expressed as the means ± S.E. in WT (n = 5), vehicle-, losartan-, and metformin-treated C57BL/6 Col4a5 G5X Alport syndrome mice (n = 8–9 per group). P values were assessed by Dunnett’s test (^##P < 0.01 vs WT. *P < 0.05, **P < 0.01 vs vehicle). (m) Whole kidney lysates were analyzed by immunoblotting. The full-length blots are presented in Supplementary Fig. S11. (n,o) The relative amount of proteins was quantified. Bars indicate the mean ± S.E. (n = 4 per group). P values were assessed by Dunnett’s test (*P < 0.05, **P < 0.01 vs vehicle).

Figure 3

Metformin regulates various intracellular signaling pathways in kidney of Col4a5 G5X Alport syndrome mice. (a) Whole kidney lysates were analyzed by immunoblotting. The full-length blots are presented in Supplementary Fig. S12. (b–h) The relative amount of proteins was quantified. Losartan or metformin decreased the level of (c) phospho-p38, (d) phospho-STAT3, (e) phospho-mTOR. Only metformin increased the level of (a) phospho-AMPK, (f) phospho-ERK, (g) phospho-p53 and (h) Nrf2. Data are expressed as the means ± S.E. (n = 4 per group). P values were assessed by Dunnett’s test (*P < 0.05, **P < 0.01 vs vehicle).

Figure 4

Transcriptome analysis reveals the comprehensive effects of metformin in kidney of Col4a5 G5X Alport syndrome mice. (a) Venn diagram shows the number of fluctuated genes in three comparisons (WT vs Alport vehicle, Alport vehicle vs Alport metformin, Alport vehicle vs Alport losartan). (b–d) Pathway analysis of fluctuated genes in Alport vehicle (b), Alport losartan (c), and Alport metformin (d). Red and blue bars represent the number of upregulated and downregulated genes, respectively. Pathways are displayed in ascending order of P value (P < 0.05). †: Common pathways between Alport/vehicle vs Alport/losartan + metformin group.

Figure 5

Transcriptome analysis reveals the comprehensive effects of metformin in glomeruli of Col4a5 G5X Alport syndrome mice. (a) Venn diagram shows the number of fluctuated genes in three comparisons (WT vs. Alport vehicle, Alport vehicle vs. Alport metformin, Alport vehicle vs Alport losartan). (b–d) Pathway analysis of fluctuated genes in the different treatment groups, as indicated. Red and blue bars represent the number of upregulated and downregulated genes, respectively. Pathways are displayed in ascending order of P value (P < 0.05). †: Common pathways between Alport vehicle vs Alport losartan + metformin group.

Figure 6

Metformin protects against podocyte dysfunction in Col4a5 G5X Alport syndrome mice. (a) Visualization of indicated podocyte and glomerular cell proteins in renal tissue sections. Scale bars, 50 μm. (b) Quantification of WT1-positive cells in the glomerulus showed losartan or metformin protected against podocyte loss in late stage C57BL/6 Col4a5 G5X vAlport syndrome mice. (c) Quantification of nephrin- and synaptopodin-positive area in the glomerulus showed that the decrease of these podocyte proteins in glomerulus was suppressed in losartan- or metformin-treated C57BL/6 Col4a5 G5X Alport syndrome mice. (d) Quantification of PCNA-positive cells in the glomerulus showed the number of proliferating cells in glomerulus was decreased in losartan- or metformin-treated C57BL/6 Col4a5 G5X Alport syndrome mice. Data are expressed as the means ± S.E. (n = 4 per group). P values were assessed by Dunnett’s test (^##P < 0.01 vs WT. *P < 0.05, **P < 0.01 vs vehicle).

Figure 7

Combining losartan and low-dose metformin extends the lifespan of Alport syndrome mice. (a) The age at ESKD of C57BL/6 background Col4a5 G5X Alport syndrome mice treated as indicated was determined. Losartan or metformin increased the age at ESKD. **P < 0.01 by Wilcoxon test (n = 9–10). (b) The age at ESKD of 129S1/SvImJ background Col4a3^−/− mice treated as indicated was determined. Low-dose metformin (2.5 mg/mL) was beneficial when combined with losartan. *P < 0.05, **P < 0.01, ***P < 0.005 by Wilcoxon test (n = 6–8).