Mesenchymal stem cells and endothelial progenitor cells decrease renal injury in experimental swine renal artery stenosis through different mechanisms

Abstract

Endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) augment tissue repair but possess slightly different properties. How the cellular phenotype affects the efficacy of this approach in renovascular disease is incompletely understood. This study tested the hypothesis that EPC and MSC protect the poststenotic kidney by blunting different disease pathways. Peripheral blood EPC and adipose-derived MSC were expanded and characterized by cell surface markers (e.g., CD34/kinase insert domain receptor, or CD44/CD90). Single-kidney hemodynamics and function were assessed in pigs after 10 weeks of renal artery stenosis (RAS) treated 4 weeks earlier with an intrarenal infusion of vehicle (n = 7), EPC (RAS+EPC) or MSC (RAS+MSC) (both 10 × 10(6), n = 6), and normal controls (n = 7). Kidney disease mechanisms were evaluated ex vivo. The ability of EPC and MSC to attenuate endoplasmic reticulum (ER) stress was also studied in isolated ER and in tubular cells cocultured with EPC and MSC. Glomerular filtration rate in RAS was lower than controls, increased in RAS+EPC, and further improved in RAS+MSC, although both improved renal blood flow similarly. EPC prominently enhanced renal growth factor expression and decreased oxidative stress, while MSC more significantly attenuated renal inflammation, ER stress, and apoptosis. Furthermore, MSC induced a greater decrease in caspase-3 and CHOP expression in cultured tubular cells through mechanisms involving cell contact. EPC and MSC achieve a comparable decrease of kidney injury in RAS by different mechanisms, although MSC elicited slightly superior improvement of renal function. These results support development of cell-based approaches for management of renovascular disease and suggest cell selection based on the underlying pathophysiology of kidney injury.

Copyright © 2012 AlphaMed Press.

Figures

Figure 1

A: Representative immunofluorescence images (20x) for surface markers of endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC). B: MSC are capable of transdifferentiating into adipocytes, osteocytes, and chondrocytes (images are 20x). C: Cytokines released from the conditioned medium of EPC and MSC. MSC released greater amounts of vascular endothelial growth factor (VEGF), but lower amounts of tumor necrosis factor (TNF)-α than EPC. *p

Figure 2

Top: Representative images of CM-DiI labeled (red) endothelial progenitor cells (EPC) or mesenchymal stem cells (MSC) in the post-stenotic kidneys of pigs with renal artery stenosis (RAS) 4 weeks after cell delivery. Green shows peanut agglutinin (PA, green arrow), a distal tubular marker, and cyan shows a proximal tubular marker phaseolus vulgaris erythroagglutinin (PHA-E, cyan arrow). EPC showed mainly tubular engraftment (yellow arrow), while MSC tend to integrate into both proximal tubules (yellow arrow) and interstitial area (red arrow). Bottom: Both EPC and MSC improved RBF and GFR in pigs with RAS, yet MSC more effectively restored GFR. *p

Figure 3

Representative micro-CT images of pig kidney samples from normal, renal artery stenosis (RAS), and RAS treated with endothelial progenitor cells (EPC) or mesenchymal stem cells (MSC). Both EPC and MSC improved microvascular density in the stenotic kidney but more effectively in the outer than inner cortex. *p

Figure 4

Western blotting images and quantification of renal angiogenic (A), oxidative and inflammatory (B), and apoptosis (C) protein expression in normal, renal artery stenosis (RAS), RAS treated with endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) pigs. (D). Endoplasmic reticulum (ER) stress assessed in ER isolated from the same kidneys. *p

Figure 5

Representative images of trichrome (blue, top 20x) and TUNEL (middle, green, arrow) staining of kidneys from normal, renal artery stenosis (RAS), RAS treated with endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) pigs. EPC and MSC similarly attenuated renal fibrosis (bottom left), but MSC more efficiently prevented renal cell apoptosis (bottom right) in the stenotic kidney. *p

Figure 6

Top: Western blotting images and quantification of the expression of CHOP and caspase-3 showing the effects of endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) on thapsigargin (TG)-induced endoplasmic reticulum (ER) stress and apoptosis in porcine kidney tubular cells in-vitro. Protection of kidney tubular cells from TG-induced ER stress and apoptosis required cell-cell contact (A) and was virtually absent when tubular cells were cultured separately and came in contact only with the culture medium (B). MSC induced greater decreases in CHOP and caspase-3 than EPC during co-incubation with tubular cells. C: immunohistochemistry of CHOP and GRP94 in kidney sections, localizing these ER stress indices to endothelial and proximal tubular cells. *p