Endothelial progenitor cells restore renal function in chronic experimental renovascular disease

Abstract

Background: Endothelial progenitor cells (EPCs) promote neovascularization and endothelial repair. Renal artery stenosis (RAS) may impair renal function by inducing intrarenal microvascular injury and remodeling. We investigated whether replenishment with EPCs would protect the renal microcirculation in chronic experimental renovascular disease.

Methods and results: Single-kidney hemodynamics and function were assessed with the use of multidetector computed tomography in vivo in pigs with RAS, pigs with RAS 4 weeks after intrarenal infusion of autologous EPCs, and controls. Renal microvascular remodeling and angiogenic pathways were investigated ex vivo with the use of micro-computed tomography, histology, and Western blotting. EPCs increased renal expression of angiogenic factors, stimulated proliferation and maturation of new vessels, and attenuated renal microvascular remodeling and fibrosis in RAS. Furthermore, EPCs normalized the blunted renal microvascular and filtration function.

Conclusions: The present study shows that a single intrarenal infusion of autologous EPCs preserved microvascular architecture and function and decreased microvascular remodeling in experimental chronic RAS. It is likely that restoration of the angiogenic cascade by autologous EPCs involved not only generation of new vessels but also acceleration of their maturation and stabilization. This contributed to preserving the blood supply, hemodynamics, and function of the RAS kidney, supporting EPCs as a promising therapeutic intervention for preserving the kidney in renovascular disease.

Figure 1

Top: Changes in blood pressure over time (n=3–4 animals per group) Bottom: Renal blood flow (RBF, a), glomerular filtration rate (GFR, b), and cortical perfusion (c) at baseline and in responses to acetylcholine in normal, renal artery stenosis (RAS), and RAS treated with autologous endothelial progenitor cells (RAS+EPC) animals. EPC infusion did not decrease blood pressure in RAS. The blunted basal and challenged renal hemodynamics and function in RAS were normalized after EPC, suggesting restoration of renovascular endothelial function. *p<0.05 vs. Normal, †p<0.05 vs. baseline, ** p<0.05 vs. Normal (two-way repeated measures ANOVA)

Figure 2

Representative immunoblots (top) and densitometric quantification (bottom) demonstrating renal protein expression of phosphorylated Akt, phosphorylated eNOS, VEGF, HIF-1α, and angiopoietin-1 in normal, kidneys with renal artery stenosis (RAS), and RAS treated with autologous endothelial progenitor cells (RAS+EPC). Replenishment of EPC in RAS augmented the expression of vascular growth factors in the stenotic kidney, suggesting a pro-angiogenic stimulus. *p<0.05 vs. Normal. †p<0.05 vs. RAS, # p=0.079 vs. Normal.

Figure 3

Representative 3D tomographic images of renal cortex and medulla (top) and quantification (bottom) from normal, renal artery stenosis (RAS), and RAS treated with autologous endothelial progenitor cells (RAS+EPC) kidneys. EPC in RAS augmented intra-renal micro-vascular density throughout the cortex, which consequently restored renal vascular volume fraction. *p<0.05 vs. Normal. †p<0.05 vs. RAS.

Figure 4

a) Representative double fluorescence of CM-DiI (red) and immunoreactivity of Oct-4 (green, x40) in frozen renal sections. b) Representative staining (x40), Western blotting bands, and densitometry (bottom) of renal immunoreactivity to Oct-4 in normal, renal artery stenosis (RAS), and RAS treated with autologous endothelial progenitor cells (RAS+EPC) kidneys. Some Oct-4+ cells are of EPC origin (dashed arrow) but others are not (white arrow), suggesting recruitment of endogenous progenitor cells. *p<0.05 vs. Normal, †p<0.05 vs. RAS.

Figure 5

a) Representative immunoblots and densitometric quantification demonstrating renal protein expression of TGF-β, MMP-2, MMP-9, and TIMP-1, and pictures showing renal immunoreactivity of α-SMA+ cells (arrows), likely myofibroblasts. b) Renal trichrome staining (x40) and quantification of renal fibrosis and glomerulosclerosis in normal, renal artery stenosis (RAS), and RAS treated with autologous endothelial progenitor cells (RAS+EPC) kidneys. EPC significantly decreased pro-fibrotic activity and attenuated renal remodeling in RAS. *p<0.05 vs. Normal, †p<0.05 vs. RAS.

Figure 6

Left: Representative immunoblots and densitometry demonstrating protein expression of TGF-β, MMP-2, MMP-9, TIMP-1, and VEGF in the contralateral kidney (CLK). Right: Renal trichrome staining (x40) of normal kidneys and CLK of renal artery stenosis (RAS) and RAS treated with endothelial progenitor cells (RAS+EPC) animals. Intra-renal infusion of EPC in the stenotic kidney only augmented VEGF in the CLK, but did not have any effect in attenuating renal fibrosis or regulating the expression of its mediators. *p<0.05 vs. Normal