GYY4137, a Hydrogen Sulfide Donor Modulates miR194-Dependent Collagen Realignment in Diabetic Kidney

Abstract

The relationship between hydrogen sulfide (H₂S), microRNAs (miRs), matrix metalloproteinases (MMPs) and poly-ADP-ribose-polymerase-1 (PARP-1) in diabetic kidney remodeling remains mostly obscured. We aimed at investigating whether alteration of miR-194-dependent MMPs and PARP-1 causes renal fibrosis in diabetes kidney, and whether H₂S ameliorates fibrosis. Wild type, diabetic Akita mice as well as mouse glomerular endothelial cells (MGECs) were used as experimental models, and GYY4137 as H₂S donor. In diabetic mice, plasma H₂S levels were decreased while ROS and expression of its modulator (ROMO1) were increased. In addition, alteration of MMPs-9, -13 and -14 expression, PARP-1, HIF1α, and increased collagen biosynthesis as well as collagen cross-linking protein, P4HA1 and PLOD2 were observed along with diminished vascular density in diabetic kidney. These changes were ameliorated by GYY4137. Further, downregulated miRNA-194 was normalized by GYY4137 in diabetic kidney. Similar results were obtained in in vitro condition. Interestingly, miR-194 mimic also diminished ROS production, and normalized ROMO1, MMPs-9, -13 and -14, and PARP-1 along with collagen biosynthesis and cross-linking protein in HG condition. We conclude that decrease H₂S diminishes miR-194, induces collagen deposition and realignment leading to fibrosis and renovascular constriction in diabetes. GYY4137 mitigates renal fibrosis in diabetes through miR-194-dependent pathway.

Figure 1

(A,B) GYY4137 treatment ameliorated increased plasma H₂S levels (A) and decreased blood glucose (B) levels in Akita mice. Plasma H₂S and blood glucose levels were measured as described in the materials and methods. Data represents mean ± SEM, n = 6–7/ group; ^†p < 0.05 vs. WT and *p < 0.05 vs. Akita. (C–F) Increased ROS and ROMO1 in diabetic kidney was mitigated by GYY4137. (C) GYY4137 decreased ROS, especially superoxide in diabetic kidney. Renal superoxide was measured by DHE fluorescence activity as described in the material and methods. Scale bar 20 µm. (D) GYY4137 also decreased reactive oxygen species modulator 1 (ROMO1), which is a mitochondrial membrane protein responsible for increasing the level of ROS production. ROMO1 mRNA (top) was measured by RT-PCR, and protein expression (bottom) by Western blotting. (E) Bar graph represents DHE fluorescence activity which was measured and compared between groups as fold change. (F) Bar graphs represent mean fold change of both mRNA and protein expression of ROMO1 using GAPDH as loading control. Data represents mean ± SEM, n = 6–7/ group; ^†p < 0.05 vs. WT and *p < 0.05 vs. Akita, compared to their respective levels of mRNA and protein expression.

Figure 2

(A–D) Increased expression and activity of MMP-9 was normalized by GYY4137 in diabetic kidney. (A) The mRNA (top) and protein (bottom) expression of MMP-9 was measured by RT-PCR and Western blotting, respectively. (B) Bar graph represents mean fold change normalized with GAPDH. (C) MMP-9 activity was measured by gelatin zymography as described in the methods. (D) Bar graph represents mean fold changes of MMP-9 activity. Data represents mean ± SEM, n = 6–7/ group; ^†p < 0.05 vs. WT and *p < 0.05 vs. Akita. (E,F) Increased expression of MMP-13 was normalized in diabetic kidney following GYY4137 treatment. (E) Expression levels of gene (top) and protein (bottom) of MMP-13 in mouse kidney was measured by RT-PCR and Western blotting, respectively. (F) Bar graph represents mean fold change normalized with GAPDH. Values are mean ± SEM, n = 6–7/group; ^†p < 0.05 vs. WT and *p < 0.05 vs. Akita, compared to their respective levels of mRNA and protein expression.

Figure 3

(A–D) Altered expression of MMP-14 and PARP1 was normalized in diabetic kidney following GYY4137 treatment. (A) The mRNA (top) and protein (bottom) expression of MMP-14 in mouse kidney was measured by RT-PCR and Western blotting, respectively. (B) Bar graph represents mean fold change of MMP-14 normalized with GAPDH. (C) The mRNA (top) and protein (bottom) expression of PARP1 in mouse kidney. (D) Bar graph represents mean fold change of PARP1 normalized with GAPDH. Values are mean ± SEM, n = 6–7/group; ^†p < 0.05 vs. WT and *p < 0.05 vs. Akita. (E,F) MMP-14 and PARP1 was inversely expressed in the glomerulus and GYY4137 normalized their expression in diabetic kidney. (E) Tissue immunostained images of MMP-14 (green; pointed with yellow arrows) and PARP1 (red; pointed with white arrows) in the mouse kidney. Cryosections were incubated overnight with primary antibody and counterstained with appropriate secondary antibodies. Representative images from n = 6–7/group. Original magnification × 60; Scale bar: 20 µm. (F) Bar graph represents mean fluorescence intensity changes (fold) vs WT control. Values are mean ± SEM, n = 6–7/group; ^†p < 0.05 vs. WT and *p < 0.05 vs. Akita, compared to their respective levels of mRNA and protein expression.

Figure 4

(A–D) Increased collagen (Col 1a and Col IV) gene and protein expression was mitigated by GYY4137 in diabetic kidney. (A) Gene expression of Col 1a and Col IV was measured by RT-PCR and (B) protein expression by Western blot analysis. (C) and (D) Bar graph represents mean fold change of Col1a and Col IV normalized with GAPDH. Values are mean ± SEM, n = 6–7/group; ^†p < 0.05 vs. WT and *p < 0.05 vs. Akita, compared to their respective levels of mRNA and protein expression. (E–I) Increased expression of HIF1α, P4HA1 and PLOD2 in diabetic kidney was normalized by GYY4137. (E) In the mouse kidney, gene expression of HIF1α, P4HA1 and PLOD2 was measured by RT-PCR, and (F) protein expression by Western blotting. (G,H) and (I) are the bar graphs representing mean fold change of HIF1α, P4HA1, and PLOD2 respectively normalized with GAPDH. Values are mean ± SEM, n = 6–7/group; †p < 0.05 vs. WT and *p < 0.05 vs. Akita, compared to their respective levels of mRNA and protein expression.

Figure 5

(A–C) Periglomerular collagen deposition was reduced in diabetic kidney treated with GYY4137. (A) Representative photomicrographs of collagen staining with picrosirius red (magnification, × 20). (B) Photomicrographs focusing glomerulus to visualize surrounding collagen (blue arrows; magnification × 60. (C) Bar graph representing total collagen area against the background, calculated as mean relative intensity in arbitrary unit, and presented as fold change compared to WT. Values are mean ± SEM, n = 6–7/group; †p < 0.05 vs. WT and *p < 0.05 vs. Akita. (D–F) GYY4137 treatment improved renal vascular perfusion in diabetic kidney. (D) Representative Barium x-ray kidney angiograms of all four experimental groups are shown here. (E) Vessel segment analysis of angiogram images for all experimental groups. Barium sulfate (50 mM) was infused via infra-renal aorta at constant pressure and time to visualize renal vascular architecture. VesSeg tool was used as described in methods for the analysis of vessel coverage. Total vessel area was calculated using ImageJ software. (F) Bar graph represents mean percent change in renal vessel coverage against the background. Values are mean ± SEM, n = 6–7/group; ^†p < 0.05 vs. WT and *p < 0.05 vs. Akita.

Figure 6

(A,B) Increased miR-194 expression in diabetic condition was normalized by GYY4137 treatment. The bar diagram represents expression of miR-194 in mouse kidney (A) and in mouse glomerular endothelial cells (MGECs) (B). The expression of miR-194 was decreased in both diabetic kidney tissue as well as in hyperglycemic (HG) conditions. The decreased levels of miR-194 in diabetic kidney was normalized with GYY4137 treatment (A), and in MGECs transfected with miR-194 mimic (B). RT-qPCR was performed to measure miR-194. WT and Akita mice were treated without or with GYY4137 as described in the methods. MGECs were transfected with miR-194 mimic and miR-194 inhibitor for 72 hours. Bar graph represents mean relative expression as fold change ± SEM, *p < 0.05 between groups; n = 6–7/ group or independent experiments. (C) Increased ROS in HG condition was diminished by either GYY4137 or miR-194 mimic treatment in MGECs. GYY4137 decreased ROS, especially superoxide in HG condition in MGECs (top panel). Similar effect was observed with miR-194 mimic treatment (bottom panel). Superoxide was measured by DHE fluorescent activity as described in the material and methods. Representative images are from n = 5 independent experiments.

Figure 7

Altered ROMO1, MMP-9, -13 and -14 mRNA and protein expression was ameliorated by miR-194 mimic in HG condition. (A) mRNA expression was measured by RT-PCR, and (B) protein expression by Western blotting. (C,D,E) and (F) Bar graphs represent mean fold changes of ROMO1, MMP-9, -13 and -14 respectively normalized with GAPDH / β-actin. Values are mean ± SEM, n = 5 independent experiments; ^†p < 0.05 vs. NG alone and *p < 0.05 vs. HG alone; compared to their respective levels of mRNA and protein expression.

Figure 8

Increased PARP-1, Col 1a and Col IV transcript and protein expression was mitigated by miR-194 mimic transfection in HG condition. (A) Gene expression of PARP-1, Col 1a and Col IV was measured by RT-PCR and (B) protein expression by Western blotting. (C,D) and (E) Bar graphs represent mean fold change of PARP-1, Col1a and Col IV respectively normalized with GAPDH / β-actin. Values are mean ± SEM, n = 5 independent experiments; †p < 0.05 vs. NG alone and *p < 0.05 vs. HG alone; compared to their respective levels of mRNA and protein expression.

Figure 9

Increased expression of HIF1α, PLOD2 and P4HA1 in HG condition was normalized by miR-194 mimic transfection. (A) The mRNA expression was measured by RT-PCR, and (B) protein expression by Western blotting. (C), (D) and (E) Bar graphs representing mean fold change of HIF1α, PLOD2 and P4HA1 respectively normalized with GAPDH / β-actin. Values are mean ± SEM, n = 5 independent experiments; †p < 0.05 vs. NG alone and *p < 0.05 vs. HG alone; compared to their respective levels of mRNA and protein expression.

Figure 10

(A) Schematics of chemical-protein interactions: STITCH 4.0. Stronger associations are represented by thicker lines. Protein-protein interactions are shown in blue, chemical-protein interactions are in green. Abbreviations: Plod2, Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 2; P4ha1, Prolyl 4-Hydroxylase Subunit Alpha 1; Col1a1, Collagen Type I Alpha 1 Chain; Col1a2, Collagen Type I Alpha 2 Chain; Col3a1, Collagen Type III Alpha 1 Chain; Col4a1, Collagen Type IV Alpha 1 Chain; Col5a1, Collagen Type V Alpha 1 Chain; Egln-1, Egl-9 Family Hypoxia Inducible Factor 1; Egln-3, Egl-9 Family Hypoxia Inducible Factor 3; Vhl, Von Hippel-Lindau tumor suppressor protein; Arnt, Aryl Hydrocarbon Receptor Nuclear Translocator; Hif1a, Hypoxia Inducible Factor 1 Alpha Subunit; Vegfa, Vascular Endothelial Growth Factor A; Crebbp, CAMP Responsive Element Binding Protein 1; Parp1, Poly(ADP-Ribose) Polymerase 1. (B) Schematic representation of central hypothesis. In diabetic kidney, diminished H₂S and decreased miRNA-194 induces increased production of ROS. Increased ROS, in turn causes imbalance of MMPs and upregulation of PARP-1. The alterations of H₂S, miR-194, ROS, MMPs and PARP-1 in diabetic kidney lead to collagen accumulation and re-alignment resulting in kidney fibrosis and renovascular constriction. GYY4137, a H₂S donor, treatment normalizes miR-194 and ROS-dependent downstream pathways of renovascular remodeling, and thus preserves renovascular architecture in diabetes.