ROCK1 Induces Endothelial-to-Mesenchymal Transition in Glomeruli to Aggravate Albuminuria in Diabetic Nephropathy - PubMed (original) (raw)

ROCK1 Induces Endothelial-to-Mesenchymal Transition in Glomeruli to Aggravate Albuminuria in Diabetic Nephropathy

Hui Peng et al. Sci Rep. 2016.

Abstract

Endothelial-to-mesenchymal transition (EndMT) can cause loss of tight junctions, which in glomeruli are associated with albuminuria. Here we evaluated the role of EndMT in the development of albuminuria in diabetic nephropathy (DN). We demonstrated that EndMT occurs in the glomerular endothelium of patients with DN, showing by a decrease in CD31 but an increase in α-SMA expression. In glomeruli of db/db mice, there was an increased ROCK1 expression in the endothelium plus a decreased CD31-positive cells. Cultured glomerular endothelial cells (GEnCs) underwent EndMT when stimulated by 30 mM glucose, and exhibited increased permeability. Meanwhile, they showed a higher ROCK1 expression and activation. Notably, inhibition of ROCK1 largely blocked EndMT and the increase in endothelial permeability under this high-glucose condition. In contrast, overexpression of ROCK1 induced these changes. Consistent alterations were observed in vivo that treating db/db mice with the ROCK1 inhibitor, fasudil, substantially suppressed the expression of α-SMA in the glomerular endothelium, and reduced albuminuria. Thus we conclude that ROCK1 is induced by high glucose and it stimulates EndMT, resulting in increased endothelial permeability. Inhibition of ROCK1 could be a therapeutic strategy for preventing glomerular endothelial dysfunction and albuminuria in developing DN.

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Figures

Figure 1

Figure 1. Evidence for EndMT in glomeruli of patients with DN and db/db diabetic mice.

(a) Periodic acid Schiff staining of healthy kidney section (left) and the kidney section from patient with DN (right). (b) Confocal microscopy reveals that mesenchymal marker (α-SMA, green) co-localized with endothelial marker (CD31, red) in glomerulus of patient with DN. (c) Corresponding higher magnification of selected area in (b). White arrows indicate that CD31-positive cells express α-SMA. (d) Periodic cid Schiff staining of the glomerulus of db/m (left) and db/db mice with early stage of DN (right). (e) Confocal microscopy reveals that α-SMA and CD31 were co-stained in glomerulus of db/db mice with early stage of DN (right). Double immunofluorescent staining of CD31 (red) and α-SMA (green). (f) Corresponding higher magnification of selected area in (e). White arrows indicate the co-localization of CD31 and α-SMA.

Figure 2

Figure 2. High glucose induces EndMT in cultured GEnCs.

GEnCs were treated with 5.5 mM glucose (normal glucose, NG) or 30 mM glucose (high glucose, HG) for 5 days. (a) mRNA levels of endothelial markers (VE-cadherin, CD31) and mesenchymal markers (α-SMA, Snail) were accessed using real-time PCR. (b) protein levels of VE-cadherin, CD31, α-SMA and Snail were accessed with immunoblotting. (c) Immunofluorescence double-staining of VE-cadherin (green) and α-SMA (red) in GEnCs treated with NG and HG. Values are mean ± SEM from 5 experiments, *P < 0.05 vs. NG (t-test). **P < 0.01 vs. NG (t-test). NG: normal glucose, HG: high glucose, VE-cdh: VE-cadherin.

Figure 3

Figure 3. High glucose mediates ROCK1 activity in glomerular endothelial cells.

(a) GEnCs were treated with NG or HG for 48 hr, the F-actin was visualized with Phalloidin - Fluor 488 (green), nuclei were stained with Propidium iodide (PI, Red). (b) GEnCs were treated with NG or HG. ROCK1 expression and activity were detected by immunoblotting. ROCK1 activity was assessed by the level of phosphorylated MYPT1 (p-MYPT1). Values are mean ± SEM from 5 experiments. *P < 0.05 vs. NG (t-test). NG: normal glucose, HG: high glucose. (c) Paraffin sections (left panel) of kidneys of db/m and db/db mice were stained with Periodic acid-Schiff (PAS). Cryosections (right panel) of kidneys of db/m and db/db mice were stained with CD31 (red) and ROCK1 (green). CD31 and ROCK1 colocalizations are indicated by arrows.

Figure 4

Figure 4. ROCK1 mediates high-glucose-induced EndMT in cultured GEnCs.

(a) GEnCs were treated with HG in the presence or absence of ROCK inhibitor Y27632 (10 μmol/L), ROCK1 activity was assessed with immunoblotting using anti-pMYPT antibody. (b) VE-cadherin and α-SMA expressions were accessed using immunoblotting. Values are mean ± SEM from 5 experiments. *P < 0.05, vs. NG , Δ_P_ < 0.05, vs. HG (two-way ANOVA, followed by Bonferroni’s multiple comparisons test). (c) Morphological characteristics were observed using immunofluorescent microscopy (400×). Red and green represented α-SMA and VE-cadherin respectively. (d) GEnCs were infected with adenovirus containing ROCK1-overexpression cassette for 2 days under NG. The ROCK1 expression and activity were examined with immunoblotting. Values are mean ± SEM from 3 experiments. *P = 0.03 vs. Ad-β-Gal (t-test). (e) mRNAs and proteins of VE-cadherin and α-SMA were determined using real-time PCR (upper panel) and immunoblotting (lower panel). Values are mean ± SEM from 3 experiments. *P < 0.05 vs. Ad-β-Gal (t-test). NG: normal glucose, HG: high glucose, VE-cdh: VE-cadherin; Ad-β-Gal: adenovirus containing β-galactosidase cassette; Ad-ROCK1: adenovirus containing ROCK1-overexpression cassette.

Figure 5

Figure 5. ROCK1 inhibition prevents high-glucose-induced hyperpermeability in GEnCs and proteinuria in mice with early stage of DN.

(a) 70% confluent GEnCs plated on transwell inserts were treated with 30 mM glucose for 5 days with or without ROCK1 inhibitor, Y27632 (10 μmol/L). Endothelial barrier function was determined by measuring trans-endothelial electrical resistance (TEER). (b) With same treatment as in (a) the permeability of GEnCs was examined by the amount of FITC-dextran across GEnCs monolayer. Values are mean ± SEM from 3 experiments. *P < 0.01, vs. NG, Δ_P_ < 0.01, vs. HG (two-way ANOVA, followed by Bonferroni’s multiple comparisons test). (c) 12-week-old db/m or db/db mice were treated with saline or fasudil (ROCK1 inhibitor, 10 mg/kg/day, intraperitoneal injection, i.p.) for 4 weeks. 24-h urine was collected and applied for SDS-PAGE followed by Coomassie brilliant blue staining. (d) The albumin excretion was accessed with urinary albumin/creatinine ratio. Values are mean ± SEM from 5 mice in each group. *P = 0.0012 vs. db/m. Δ_P_ = 0.0231, vs. db/db (two-way ANOVA, followed by Bonferroni’s multiple comparisons test). NG: normal glucose, HG: high glucose. (e) Cryosections of kidneys (from mice described in (c) were subjected to double immunofluorescent staining with CD31 (red) and p-MYPT1 (green). Arrows indicate p-MYPT1 staining in CD31-positive cells.

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