Liraglutide Reduces Both Atherosclerosis and Kidney Inflammation in Moderately Uremic LDLr-/- Mice

Abstract

Chronic kidney disease (CKD) leads to uremia. CKD is characterized by a gradual increase in kidney fibrosis and loss of kidney function, which is associated with a progressive increase in risk of atherosclerosis and cardiovascular death. To prevent progression of both kidney fibrosis and atherosclerosis in uremic settings, insight into new treatment options with effects on both parameters is warranted. The GLP-1 analogue liraglutide improves glucose homeostasis, and is approved for treatment of type 2 diabetes. Animal studies suggest that GLP-1 also dampens inflammation and atherosclerosis. Our aim was to examine effects of liraglutide on kidney fibrosis and atherosclerosis in a mouse model of moderate uremia (5/6 nephrectomy (NX)). Uremic (n = 29) and sham-operated (n = 14) atherosclerosis-prone low density lipoprotein receptor knockout mice were treated with liraglutide (1000 μg/kg, s.c. once daily) or vehicle for 13 weeks. As expected, uremia increased aortic atherosclerosis. In the remnant kidneys from NX mice, flow cytometry revealed an increase in the number of monocyte-like cells (CD68+F4/80-), CD4+, and CD8+ T-cells, suggesting that moderate uremia induced kidney inflammation. Furthermore, markers of fibrosis (i.e. Col1a1 and Col3a1) were upregulated, and histological examinations showed increased glomerular diameter in NX mice. Importantly, liraglutide treatment attenuated atherosclerosis (~40%, p < 0.05) and reduced kidney inflammation in NX mice. There was no effect of liraglutide on expression of fibrosis markers and/or kidney histology. This study suggests that liraglutide has beneficial effects in a mouse model of moderate uremia by reducing atherosclerosis and attenuating kidney inflammation.

Fig 1. Uremia accelerates atherosclerosis in LDLr-/- mice.

Uremic (NX; n = 10) and control (SHAM; n = 10) LDLr-/- mice were put on a western type diet for 9 weeks after induction of uremia. Atherosclerosis was determined as the relative plaque area in % of the total aortic arch area. Depicted values are mean±SEM. *p<0.05 as determined by unpaired students t-test.

Fig 2. Immune cell composition is changed in kidneys from uremic LDLr-/- mice.

Sixteen weeks after induction of uremia, flow cytometry was performed on kidneys from control (SHAM) and uremic (NX) LDLr-/- mice (n = 5 mice/group). The number of CD4⁺ T-cells (A), CD8⁺ T-cells (B), monocyte-like (CD68⁺F4/80^-) and macrophage-like (CD68⁺F4/80⁺) cells (C and D) relative to kidney weight is depicted. On macrophage-like cells, median fluorescent intensity (MFI) for the M1 marker CD11c (E) or the M2 marker CD206 (F) was detected. Depicted values are mean±SEM. *p<0.05 as determined by unpaired students t-test.

Fig 3. Liraglutide attenuates atherosclerosis in uremic LDLr-/- mice.

Uremic LDLr-/- mice were treated with vehicle (NX; n = 14) or liraglutide (NX LIRA; n = 15) and sham operated control LDLr-/-mice were treated with vehicle (SHAM; n = 14). After 11 weeks of treatment with full dose (1000 μg/kg), atherosclerosis was quantified as the relative plaque area in % of the total aortic arch area in all mice (A) and in NX mice with urea levels >20 (B). Depicted values are mean±SEM. *p<0.05 as determined by 1-way ANOVA followed by Sidak’s multiple comparisons post-test. n = 14–15 mice per group.

Fig 4. Liraglutide attenuates NX mediated kidney inflammation in LDLr-/- mice.

A-F: flow cytometry analysis of kidneys from control (SHAM; n = 7), uremic (NX; n = 7) or liraglutide treated uremic (NX LIRA; n = 7) LDLr-/- mice showing the number of CD4⁺ T-cells (A), CD8⁺ T-cells (B), monocyte-like (CD68⁺F4/80^-) and macrophage-like (CD68⁺F4/80⁺) cells (C and D) relative to kidney weight. On macrophage-like cells, median fluorescent intensity (MFI) for the M1 marker CD11c (E) or the M2 marker CD206 (F) was detected. Depicted values are mean±SEM. *p<0.05, **p<0.01, ***p<0.005 as determined by 1-way ANOVA followed by Sidak’s multiple comparisons post-test. n = 6 mice per group.

Fig 5. Uremia increases glomerular size, but not cortical collagen content in LDLr-/- mice.

Representative pictures of kidney sections from control (SHAM), uremic (NX) and liraglutide treated uremic (NX LIRA) mice stained with Massons trichrome (A). Scale bar = 200 μm for top row and 100 μm for bottom row. Glomerular diameters were measured (B) (25–49 glomeruli were assessed per kidney, n = 7–8 in each group) and collagen deposition in the kidney cortex was quantified (C) (n = 5–7 in each group) using the Visiopharm software. **p<0.01, as determined by 1-way ANOVA followed by Sidak’s multiple comparisons post-test.