Xiao-Shen-Formula, a Traditional Chinese Medicine, Improves Glomerular Hyper-Filtration in Diabetic Nephropathy via Inhibiting Arginase Activation and Heparanase Expression

Abstract

Hyperglycemia induces glomerular hyper-filtration, which contributes to the development of diabetic nephropathy (DN), a condition that remains a challenge for treatment. The present study investigated the effect of Xiao-Shen-Formula (XSF) used for treatment of renal injury in type 1 DN mice model induced by streptozotocin (STZ) and its underlying mechanism in cultured human glomerular endothelial cell (hGECs). Studies were performed using control, diabetic DN, DN treated with XSF groups (1 g/kg/d, LXSF or 3 g/kg/d, HXSF) for 6 weeks and hGECs were post-treated with mice serum containing HXSF (MS-HXSF) and arginase inhibitor (ABH, 100 μM) in high glucose medium. HXSF treatment restored STZ-induced renal hyper-filtration, glomerulosclerosis, renal microvascular remodeling and the increased levels of systemic reactive oxidative species and inflammatory cytokines, accompanied by preventing the decreased expression of glomerular heparin sulfate and the increased levels of cortical heparanase and argianse2 protein and arginase activity. In hGECs study, MS-HXSF ameliorated the enhancement in arginase activity, the protein/mRNA expression of heparanase, mRNA levels of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, monocyte chemoattractant protein-1 and permeability of hGECs monolayers as well as the depression of nitric oxide production. Besides all these protective effects, XSF blunted the mRNA expression of TNF-α in vivo and vitro studies as well, which was not changed by the post-treatment of ABH or HXSF plus ABH. This study demonstrated that the protective effect of XSF might be related with vascular prevention, anti-inflammation and anti-oxidation through intervening multi-targets including glomerular endothelial arginase-heparanase signaling pathway in DN model.

Keywords: Xiao-Shen-Formula; arginase; diabetic nephropathy; glomerular hyper-filtration; heparanase; nitric oxide.

FIGURE 1

Xiao-Shen-Formula (XSF) ameliorated proteinuria and kidney impairments in DN mice. (A) The urinary microalbuminuria and creatinine ratio (mAlb/Cr), (B) 24 h-UTP and (C) creatinine clearance (CCr) were measured at 6 weeks after XSF treatment. Control: non-diabetic normal mice; DN: diabetic nephropathy; LXSF and HXSF: DN mice were treated with 1 or 3 g/kg/d XSF respectively. Data are presented as mean ± SE. ^∗P < 0.05 vs. Control, ^#P < 0.05 vs. DN, n = 10 mice/group.

FIGURE 2

XSF prevented the damaged of glomerular morphology in DN mice. (A) Light microscopy of renal mesangial regions stained by PAS. Scale bar: 20 μm. (B) Quantitation of PAS staining were shown by PAS staining score. Data are presented as mean ± SE. ^∗P < 0.05 vs. Control, ^#P < 0.05 vs. DN, n = 6 mice/group.

FIGURE 3

XSF prevented the impairment of microvascular remodeling renal cortical α-smooth muscle actin (α-SMA) density in DN mice. (A) α-SMA immunolocalization (brown staining). Scale bar: 50 μm. (B) Quantification of α-SMA density. Data are presented as mean ± SE. ^∗P < 0.05, n = 5 mice/group.

FIGURE 4

XSF blunted the elevation of reactive oxidative species and cytokines in DN mice. (A) Effect of XSF on plasma malonaldehyde (MDA). (B) Effect of XSF on plasma cytokines including proinflammatory cytokines tumor necrosis factor α (TNF-α) and IL-6 and anti-inflammatory IL-10. Data are presented as mean ± SE. ^∗P < 0.05 vs. Control, ^#P < 0.05 vs. DN, n = 5 mice/group.

FIGURE 5

XSF inhibited cortical heparanase expression and restored the decreased glomerular heparan sulfate (HS) contents in DN mice. Effect of XSF on (A,B) protein and mRNA of heparanase; (C) the glomerular heparanase expression was determined by immunofluorescence staining. Scale bar, 20 μm. (D) The columns quantified the amount of immuno-reactive heparanase by NIH Image software. (E) The glomerular HS expression was determined by immunofluorescence staining. (a) Control; (b) DN; (c) LXSF; (d) HXSF. Scale bar, 20 μm. (F) The columns quantified the amount of immuno-reactive HS by NIH Image software. Data are presented as mean ± SE. ^∗P < 0.05 vs. Control, ^#P < 0.05 vs. DN, n = 5–10 mice/group.

FIGURE 6

XSF attenuated plasma and cortical arginase activity and cortical arginase 2 (ARG2) expression. (A) Effect of XSF on plasma arginase activity, (B) cortical argianse activity, and (C) cortical ARG2 expression. (D) The glomerular ARG2 expression was determined by immunofluorescence staining. Scale bar: 20 μm. (E) The columns quantified the amount of immuno-reactive ARG2 by NIH Image software. Data are presented as mean ± SE. ^∗P < 0.05, n = 5 mice/group.

FIGURE 7

Effect of XSF on arginase-NO-heparanse pathway in hGECs. Effect of XSF (3 g/kg/d) containing serum (HSXF) on (A) arginase activity, (B) NO production, and (C,D) heparanse mRNA/protein in human glomerular endothelial cells (hGECs) with and without arginase inhibitor (ABH, 100 mM) under 24 h exposing to normal glucose (NG, 5 mM) and high glucose (HG, 25 mM). BS: basal serum. Data are presented as mean ± SE. ^∗P < 0.05 vs. Control, ^#P < 0.05 vs. DN, n = 5 experiments.

FIGURE 8

Effect of HXSF containing serum on the permeability of hGECs and mRNA of cytokine, chemokine, and adhesion molecules in hGECs. (A) The levels of proinflammatory cytokines TNF-α, the chemokine monocyte chemoattractant protein-1 (MCP-1), and the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule 1 (ICALM-1). (B) Transendothelial passage of FITC-dextran was used to determine permeability of hGECs monolayer, seeded onto collagen-coated transwells. Fluorescent density of samples was analyzed on a paradigm microplate fluorometer. BS, basal serum. Data are presented as mean ± SE. ^∗P < 0.05 vs. Control, ^#P < 0.05 vs. DN, n = 5 experiments.

FIGURE 9

Schematic diagram representing XSF on amelioration of heparanase expression and improvement of glomerular hyperpermeability by inhibtion of arginase activation.