MMP-9- and NMDA receptor-mediated mechanism of diabetic renovascular remodeling and kidney dysfunction: hydrogen sulfide is a key modulator

Abstract

Previously we reported that matrix metalloproteinase-9 (MMP-9) plays an important role in extracellular matrix (ECM) remodeling in diabetic kidney. Induction of NMDA-R and dysregulation of connexins (Cxs) were also observed. We concluded that this was due to decreased H2S production by downregulation of CBS and CSE enzymes. However, the potential role of H2S to mitigate ECM dysregulation and renal dysfunction was not clearly understood. The present study was undertaken to determine whether H2S supplementation reduces MMP-9-induced ECM remodeling and dysfunction in diabetic kidney. Wild type (C57BL/6J), diabetic (Akita, C57BL/6J-Ins2(Akita)), MMP-9 knockout (MMP-9(-/-), M9KO) and double KO of Akita/MMP-9(-/-) (DKO) mice were treated without or with 0.005 g/l of NaHS (as a source of H2S) in drinking water for 30 days. Decreased tissue production and plasma content of H2S in Akita mice were ameliorated with H2S supplementation. Dysregulated expression of MMP-9, CBS, CSE, NMDA-R1 and Cxs-40, -43 was also normalized in Akita mice treated with H2S. In addition, increased renovascular resistive index (RI), ECM deposition, plasma creatinine, and diminished renal vascular density and cortical blood flow in Akita mice were normalized with H2S treatment. We conclude that diminished H2S production in renal tissue and plasma levels in diabetes mediates adverse renal remodeling, and H2S therapy improves renal function through MMP-9- and NMDA-R1-mediated pathway.

Keywords: Collagen–elastin; Connexins; Diabetes; Extracellular matrix; Hydrogen sulfide; Matrix metalloproteinase; Nephropathy; Renal microvasculature.

Fig. 1. NaHS treatment increased tissue production and plasma levels of H₂S in diabetic animals

Animals were treated without or NaHS (0.05 g/ L) for 30 days as mentioned in Materials and Methods. (A) Fifty milligram (50 mg) of kidney tissue from each animal group was homogenized and assayed for H₂S measurement as described in Materials and Methods. (B) Hundred microliter of plasma from each animal was used for H₂S content measurement. H₂S measurement for both tissue and plasma was performed within 36 h of sample collection. Data represents mean ± SEM, (n = 8).

Fig. 2. Increased expression and activity of MMP-9 in diabetes was by H₂S

Kidneys were homogenized in appropriate buffer, protein extracted and loaded onto SDS gel as described in Materials and Methods. (A) MMP-9 protein expression. GAPDH was used as loading control. (B) Gelatin zymography image showing MMP-9 activity. (C) Fold changes of respective proteins. (D) Bar diagram represents fold change of the protein and enzyme activity. Data represents mean ± SEM from seven independent experiments (n = 8).

Fig. 3. Decreased expression and localization of CBS & CSE were normalized by H₂S in diabetic mice

Kidneys were homogenized in RIPA buffer and loaded onto SDS gel as described in Materials and Methods. (A) Protein expression of CBS and CSE. GAPDH was used as loading control. (B) Bar diagram represents fold changes of CBS and CSE expression. Data represents mean ± SEM from five independent experiments (n = 8). (C and D) Immunostaining images of CBS (green; pointed with red arrow) and CSE (red; pointed with yellow arrow) enzymes in kidney tissues.

Fig. 4. Up regulated NMDA-R1, Cx-40 and -43 in diabetic animals were normalized with H₂S treatment

200 μg of kidney tissue protein were loaded onto SDS gel and immunoblotted as described in Materials and Methods. (A) Expression of NMDA-R1, Cx-40 and -43 proteins without or with H₂S treatment. GAPDH was used as control. (B) Fold changes of respective proteins. Bar graphs represent mean ± SEM from five independent experiments (n = 8).

Fig. 5. Renal resistive index and cortical blood flow measurement. (A) Renal resistive index was reduced in diabetic animals following H₂S treatment

Kidney ultrasound was performed to measure renal resistive index (RI) using the Visual sonics Vevo Ultra imaging system as described in Materials and Methods. Values are represented as RI. Resistance of renal arterial flow to the kidney was measured by RI and calculated as (peak systolic velocity – end-diastolic velocity)/peak systolic velocity. Median values are shown in the “box-and-whisker plot”. Box plots represent mean RI ± SEM (n=5). (B) Renal cortical blood flow was increased significantly following H₂S treatment in diabetic mice. Renal cortical blood flow was measured in the left kidney as described in Materials and Methods. Box plots represent mean flux units in the renal cortex ± SEM (n=5).

Fig. 6. H₂S treatment increased renal vascular perfusion in diabetic mice

To visualize renal vascular architecture, 50 mM of barium sulfate was infused via infra-renal aorta at constant pressure and time. (A) Representative Barium x-ray kidney angiogram of WT mice without H₂S treatment is shown here as an example (left); FOV, field of view. Analysis of its vessel coverage using VesSeg tool as described in Materials and Methods is shown on the right. (B) Vessel segment analysis of angiogram images from WT, AKITA, M9KO and DKO groups without or with H₂S treatment. Total vessel area was calculated using ImageJ software. (C) Bar diagram represents the mean percent change in renal vessel coverage against the background ± SEM (n = 8 / group). (D) Representative time course angiography of Akita kidney without or with H₂S treatment.

Fig. 7. H₂S reduced increased deposition of collagen in diabetic kidney

(A) Representative photomicrographs of collagen staining with picrosirius red. Blue arrows indicated peri-glomerular and green arrows indicated tubulointerstitial collagen staining (original image × 25 magnifications). (B) Bar diagram showed total collagen area against the background presented as mean relative intensity in arbitrary unit (A.U.). Data represents mean ± SEM (n=5/group).

Fig. 8. H₂S treatment increased elastin expression in diabetic animals

(A) Kidney sections were stained with Van Gieson's stain and dark brown in the photomicrographs, indicated by blue arrows, depicted elastin in the renal perivascular region (original image × 25 magnifications). (B) Bar diagram represents the total elastin area against the background presented as mean relative intensity ± SEM (n= 5/ group).

Fig. 9. Increased plasma levels of creatinine were reduced significantly after H₂S treatment in diabetic animals

End-point plasma creatinine levels in WT, Akita, M9KO and DKO mice were measured before and after H₂S treatment. Each experiment was performed in triplicate and values presented as mean ± SEM (n=5/group).

Fig. 10. STITCH network connecting H₂S machinery, ECM and GAP junction protein

For eight different compounds, investigated in this study, interacting proteins and chemicals are shown. Stronger associations are represented by thicker lines. Protein-protein interactions are shown in blue, chemical-protein interactions are shown in green. CBS: cystathionine β-synthase; CTH: cystathionine γ-lyase; MPST: 3-mercaptopyruvate sulfurtransferase; MMP-9: Matrix metalloproteinase 9; TIMP1: Tissue inhibitor of metalloproteinase 1; Eln: Elastin; Gja1: Connexin-43; Gja5: Connexin-40; Cdh2: Cadherin 2; Prkcc: protein kinase C γ.