Reno-Protective Effect of GLP-1 Receptor Agonists in Type1 Diabetes: Dual Action on TRPC6 and NADPH Oxidases

Abstract

Diabetic kidney disease (DKD), a serious diabetic complication, results in podocyte loss and proteinuria through NADPH oxidases (NOX)-mediated ROS production. DUOX1 and 2 are NOX enzymes that require calcium for their activation which enters renal cells through the pivotal TRPC channels. Hypoglycemic drugs such as liraglutide can interfere with this deleterious mechanism imparting reno-protection. Herein, we aim to investigate the reno-protective effect of GLP1 receptor agonist (GLP1-RA), via its effect on TRPC6 and NADPH oxidases. To achieve our aim, control or STZ-induced T1DM Sprague-Dawley rats were used. Rats were treated with liraglutide, metformin, or their combination. Functional, histological, and molecular parameters of the kidneys were assessed. Our results show that treatment with liraglutide, metformin or their combination ameliorates DKD by rectifying renal function tests and protecting against fibrosis paralleled by restored mRNA levels of nephrin, DUOX1 and 2, and reduced ROS production. Treatment with liraglutide reduces TRPC6 expression, while metformin treatment shows no effect. Furthermore, TRPC6 was found to be directly interacting with nephrin, and indirectly interacting with DUOX1, DUOX2 and GLP1-R. Our findings suggest that treatment with liraglutide may prevent the progression of diabetic nephropathy by modulating the crosstalk between TRPC6 and NADPH oxidases.

Keywords: DUOX1/2; GLP-1; TRPC6; diabetic kidney disease; liraglutide.

Figure 1

Effect of the GLP1-RA, liraglutide, the AMPK activator, metformin or their combination on kidney injury. (A,B) Bar plots representing SCr and BUN concentrations respectively. (C) Bar plot reflecting the quantification of proteinuria. (D,E) Bar plots representing the glomerulosclerotic and tubulointerstitial fibrosis index (%) respectively. (H,I) Representative microphotographies of glomerular and tubular PAS stain respectively with black arrows pointing to the injury (magnification 40×, scale bar 50 µm). (F,G) Bar plots representing the quantification of the glomerular and tubular collagen deposition (%). (J,K) Representative microphotographies of glomerular and tubular MT stain with black arrows pointing to the collagen deposition (Magnification 40×, scale bar 50 µm). (L,M) Bar plots representing the differential expression of fibronectin and nephrin mRNA expression normalized against GAPDH expression respectively. GAPDH: glyceraldehyde 3-phosphate dehydrogenase. All data are expressed as mean ± SEM. * p ≤ 0.05 diabetic vs. control groups. # p ≤ 0.05 treated vs. diabetic groups.

Figure 2

AMPK mRNA levels, AMPK activity assay and GLP-1R protein expression in kidney tissues. (A) Bar plot representing the differential expression of AMPK in all rat groups normalized against GAPDH expression. (B) Bar plot representing the differential AMPK activity in all rat groups. (C) Representative immunoblot of GLP-1R protein (up), and bar plot reflecting the quantification of GLP-1R protein expression (below) against HSC70. AMPK: adenosine monophosphate kinase, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, GLP-1R: glucagon-like peptide-1 receptor, HSC70: heat shock cognate. All data are expressed as mean ± SEM. * p ≤ 0.05 diabetic vs. control groups. # p ≤ 0.05 treated vs. diabetic groups.

Figure 3

ROS production, NADPH oxidase activity and DUOX1/DUOX2 mRNA expression in the kidneys. (A) Bar plot reflecting ROS production through EOH/DHE ratio quantification. (B) Bar plot reflecting NADPH oxidase activity. (C,D) Bar plots representing the differential expression of DUOX1 and DUOX2 normalized against GAPDH expression respectively. GAPDH: glyceraldehyde 3-phosphate dehydrogenase. All data are expressed as mean ± SEM. * p ≤ 0.05 diabetic vs. control groups. # p ≤ 0.05 treated vs. diabetic groups.

Figure 4

TRPC6 protein expression in the kidneys. (A) Representatives immunoblot of TRPC6 protein (up) and bar plot representing the quantification of TRPC6 expression against HSC70. (B) Representative microphotographies of the immunohistochemistry staining of TRPC6 as indicated by the black arrows (Magnification 40X, scale bar 50 µm). HSC-70: Heat shock cognate. All data are expressed as mean ± SEM. * p ≤ 0.05 diabetic vs. control groups. # p ≤ 0.05 treated vs. diabetic groups.

Figure 5

Protein interactions between TRPC6, DUOX1, DUOX2, GLP1-R, nephrin, podocin and other proteins in humans obtained from GeneMANIA database. Colors of the matching lines in the figure indicate different types of interactions: orange line indicates predicted interaction, blue line indicates shared pathway, green indicates genetic interactions, yellow indicates shared protein domains, pink indicates physical interactions, blue indicates co-localization and purple indicates co-expression. The width of the line reflects the significance of interaction: the thicker the line, the more robust the prediction of the interaction. TRPC: transient receptor potential channel, NPHS2: podocin, NPHS1: nephrin, RNF24: RING finger protein 24, KIRREL: Kin of IRRE-like protein 1, PLCG1: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1.

Figure 6

Graphical abstract. The reno-protective effect of liraglutide is mediated by the inhibition of TRPC6/NADPH oxidases-DUOX1 and 2-ROS production signaling pathways. However, metformin reno-protective effect is mediated by damping NADPH oxidases-DUOX1 and 2-ROS production axis through AMPK activation independently from TRPC6.