Soluble thrombomodulin reduces inflammation and prevents microalbuminuria induced by chronic endothelial activation in transgenic mice

Abstract

Chronic kidney disease pathogenesis involves both tubular and vascular injuries. Despite abundant investigations to identify the risk factors, the involvement of chronic endothelial dysfunction in developing nephropathies is insufficiently explored. Previously, soluble thrombomodulin (sTM), a cofactor in the activation of protein C, has been shown to protect endothelial function in models of acute kidney injury. In this study, the role for sTM in treating chronic kidney disease was explored by employing a mouse model of chronic vascular activation using endothelial-specific TNF-α-expressing (tie2-TNF) mice. Analysis of kidneys from these mice after 3 mo showed no apparent phenotype, whereas 6-mo-old mice demonstrated infiltration of CD45-positive leukocytes accompanied by upregulated gene expression of inflammatory chemokines, markers of kidney injury, and albuminuria. Intervention with murine sTM with biweekly subcutaneous injections during this window of disease development between months 3 and 6 prevented the development of kidney pathology. To better understand the mechanisms of these findings, we determined whether sTM could also prevent chronic endothelial cell activation in vitro. Indeed, treatment with sTM normalized increased chemokines, adhesion molecule expression, and reduced transmigration of monocytes in continuously activated TNF-expressing endothelial cells. Our results suggest that vascular inflammation associated with vulnerable endothelium can contribute to loss in renal function as suggested by the tie2-TNF mice, a unique model for studying the role of vascular activation and inflammation in chronic kidney disease. Furthermore, the ability to restore the endothelial balance by exogenous administration of sTM via downregulation of specific adhesion molecules and chemokines suggests a potential for therapeutic intervention in kidney disease associated with chronic inflammation.

Fig. 1.

Continuous activation of endothelium in tie2-TNF transgenic mice induces markers of nephropathy. Both albumin normalized to creatinine levels from urine samples (A) and interstitial glomerular fibrosis from trichrome-stained kidney sections (B) were assessed in wild-type (Wt) or tie2-TNF (TNF) mice. Quantification of glomerular thickness scores are shown in C. Data shown are an average from a group size of n = 8. Scale bar = 100 μm.

Fig. 2.

Endothelial location of ICAM-1 in kidneys from tie2-TNF mice. Kidney sections from perfusion-fixed animals of tie2-TNF or wild-type background were incubated with monoclonal antibody anti-platelet endothelial cell adhesion molecule (PECAM; red) and for colocalization with monoclonal anti-ICAM-1 antibody (green) and observed by confocal microscopy. A: wild-type ICAM-1. B: tie2-TNF ICAM-1. C: tie2-TNF PECAM; D: tie2-TNF IgG background. E: tie2-TNF ICAM-1 and PECAM overlay. F: enlargement of inset in E. Scale bars = 50 μm except 100 μm in F. Data shown are from a representative experiment performed with a group size of n = 8 with similar results.

Fig. 3.

Amelioration of leukocyte extravasation in tie2-TNF mice after soluble thrombomodulin (sTM) treatment. Kidney sections from wild-type (A and C) or tie2-TNF (B and D) mice without (A and B) or with sTM treatment (C and D) were prepared and stained with a rat anti-mouse CD45 antibody using the AEC chromogen method. CD45-positive cells were quantified in a double-blinded fashion from at least 125 paraffin sections (E) representing n = 8/group. Scale bars = 20 μm.

Fig. 4.

Increased reactivity to thrombin in tie2-TNF mice. tie-2 TNF mice (age 3 mo) were tested for vascular permeability by injecting FITC-BSA into the tail vein followed by interstitial local injection of thrombin (100 U) into one ear and saline into the other. Dye was isolated from ears, and the fluorescence intensity ratio between thrombin- and saline-treated ears was employed to measure fold-induction of vascular permeability. Data shown are an average from a group size of n = 8.

Fig. 5.

Amelioration of albuminuria and fibrosis in sTM-treated tie2-TNF mice. Both albumin normalized to creatinine levels from urine samples (A) and interstitial glomerular fibrosis from trichrome-stained kidney sections (B) were assessed in tie2-TNF (TNF) or Wt mice without and with sTM treatment. ACR, albumin/creatinine ratio. Glomerular thickness scores (11) are shown in C. Data shown are an average from a group size of n = 8. Scale bars = 100 μm.

Fig. 6.

Profile of nephropathy and proinflammatory genes in kidney sections of sTM-treated tie2-TNF mice. A: heat map of kidney disease- and inflammation-relevant genes altered in tie2-TNF mice vs. wild-type mice without or with sTM treatment. B: downregulation of representative genes implicated in inflammation (VCAM-1, IL-18) and kidney remodeling [transforming growth factor-β receptor 2 (TGFβR2)] are shown.

Fig. 7.

Normalization proinflammatory molecules in sTM-treated TNF endothelial cells. A: quantitative real-time PCR analysis of inflammatory gene transcripts in control (Ctrl-EC) and activated endothelial cells (TNF-EC) without and with sTM treatment was determined. Data are shown as normalized mRNA fold-change to control cells. B: Western blot analysis for RANTES. Immunoblot analysis was performed as described in materials and methods with anti-RANTES antibody, stripped, and reprobed with GAPDH antibodies. Quantification of fold-change in protein expression normalized to GAPDH from 3 different experiments was analyzed by National Institutes of Health Image J densitometry analysis software. Data shown are from a representative experiment and repeated at least 3 additional times with similar results.

Fig. 8.

Amelioration of increased migration of leukocytes across continuously activated endothelial cells dependent on cooperation of chemokine signaling and vascular adhesion molecules by sTM. A: increased migration of THP-1 monocytic cells across TNF-expressing endothelial cells can be abrogated by pretreatment of monocytes with pertussis toxin (PTX). B: increased migration of THP-1 monocytic cells across TNF-expressing endothelial cells can be blocked by pretreatment with neutralizing antibodies to ICAM-1 and VCAM-1. C: amelioration of increased migration of monocytes across activated endothelium without and with sTM treatment is shown. D: transmigration of monocytes across activated endothelium is independent of hirudin. The data presented are from a representative experiment performed in triplicate, repeated 4 times independently with similar results.