Active maintenance of endothelial cells prevents kidney fibrosis

Abstract

Background: Soluble epoxide hydrolase (sEH) expressed by endothelial cells catalyzes the metabolism of epoxyeicosatrienoic acids (EETs), which are vasoactive agents.

Methods: We used a unilateral ureteral obstruction mouse model of kidney fibrosis to determine whether inhibition of sEH activity reduces fibrosis, the final common pathway for chronic kidney disease.

Results: sEH activity was inhibited by continuous release of the inhibitor 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) for 1 or 2 weeks. Treatment with AUDA significantly ameliorated tubulointerstitial fibrosis by reducing fibroblast mobilization and enhancing endothelial cell activity. In an in vitro model of endothelial-to-mesenchymal transition (EndMT) using human vascular endothelial cells (HUVECs), AUDA prevented the morphologic changes associated with EndMT and reduced expression of fibroblast-specific protein 1. Furthermore, HUVECs activated by AUDA prevented the epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells in a co-culture system.

Conclusion: Our findings suggest that regulation of sEH is a potential target for therapies aimed at delaying the progression of kidney fibrosis by inhibiting EndMT and EMT.

Keywords: Endothelial dysfunction; Endothelial-to-mesenchymal transition; Epithelial-mesenchymal transition; Kidney fibrosis; Soluble epoxide hydrolase.

Figure 1. Effects of soluble epoxide hydrolase (sEH) inhibition on kidney tubulointerstitial fibrosis

(A, B) Significantly reduced interstitial fibrosis following treatment with 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) in a unilateral ureteral obstruction (UUO) model. (C–E) Immunofluorescence analysis. AUDA significantly reduced the level of fibroblast-specific protein 1 (FSP-1) positive fibrosis, the proportion of FSP-1/CD31-positive cells, and preserved the peritubular capillary network. Arrows indicate FSP-1/CD3 double-positive cells. (F–H) AUDA also reduced the level of αSMA-positive fibrosis and the proportion of αSMA/CD31 double-positive cells. (I, J) Inhibition of sEH down-regulated expression of FSP-1 as demonstrated by real-time PCR (I) and Western blotting (J). Values are given as mean ± standard error of mean (n = 6 per group for each experiment). *P < 0.05, **P < 0.01, ***P < 0.001; by ANOVA test.

Figure 2. Alteration of the microenvironment by soluble epoxide hydrolase inhibition in a unilateral ureteral obstruction (UUO) model

12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) treatment suppressed transforming growth factor (TGF)-β1 and TGF-β2 (A) and increased expression of angiogenic molecules such as von Willebrand factor (vWF), CD31, and VE-cadherin (B). Values are given as mean ± standard error of mean (n = 6 per group for each experiment). *P < 0.05, **P < 0.01; by ANOVA test.

Figure 3. Regulation of endothelial-to-mesenchymal transition by soluble epoxide hydrolase inhibition in a unilateral ureteral obstruction (UUO) model

(A) Flow-cytometric analysis. The proportion of fibroblast-specific protein 1 (FSP-1)/CD31 double-positive cells among all FSP-1–positive cells was reduced by 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) treatment. (B) Immunofluorescence analyses of CD31 cells from isolated intrarenal mononuclear cells obtained from cytospin preparations. AUDA treatment reduced the proportion of von Willebrand factor/FSP-1 double-positive cells.

Figure 4. Regulation of endothelial-to-mesenchymal transition by soluble epoxide hydrolase inhibition in an in vitro model using human umbilical vein endothelial cells (HUVECs)

(A, B) Recombinant transforming growth factor (rTGF)-β2–induced fibroblast-like changes and fibroblast-specific protein 1 (FSP-1) expression in HUVECs were reversed by 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) treatment. (C–E) AUDA treatment increased expression of CD31 and reduced expression of αSMA and FSP-1. All values are given as mean ± standard error of mean (n = 4 per group for each experiment). *P < 0.05, **P < 0.01, ***P < 0.001; by ANOVA test.

Figure 5. Direct effect of soluble epoxide hydrolase inhibition on epithelial-to-mesenchymal transition (EMT) in an in vitro model using human kidney tubular epithelial cells (TECs)

(A–D) Recombinant transforming growth factor (rTGF)-β2–induced EMT of TECs was not reversed by 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) treatment. FSP-1, fibroblast-specific protein 1. *P < 0.05, **P < 0.01, ***P < 0.001; by ANOVA test.

Figure 6. Indirect effect of soluble epoxide hydrolase inhibition on epithelial-to-mesenchymal transition (EMT) in an in vitro co-culture model of human kidney tubular epithelial cells (hTECs) and human umbilical vein endothelial cells (HUVECs)

(A) Co-culture of TECs and HUVECs using the Transwell^TM system. (B–D) Activation of HUVECs by 12-(3-adamantan-1-ylureido)-dodecanoic acid treatment reversed recombinant transforming growth factor (rTGF)-β2-induced fibroblast-like changes and expression of FSP-1, αSMA, and collagen-4 in TECs. (E) The concentration of 14,15-EET-epoxyeicosatrienoic acids (EETs) in culture supernatants. Values are given as mean ± standard error of mean (n = 4 per group for each experiment). AUDA, 12-(3-adamantan-1-ylureido)-dodecanoic acid. *P < 0.05, **P < 0.01, ***P < 0.001; by ANOVA test.