Adjunctive mesenchymal stem/stromal cells augment microvascular function in poststenotic kidneys treated with low-energy shockwave therapy

Abstract

Effective therapeutic strategies are needed to preserve renal function in patients with atherosclerotic renal artery stenosis (ARAS). Low-energy shockwave therapy (SW) and adipose tissue-derived mesenchymal stem/stromal cells (MSCs) both stimulate angiogenesis repair of stenotic kidney injury. This study tested the hypothesis that intrarenal delivery of adipose tissue-derived MSCs would enhance the capability of SW to preserve stenotic kidney function and structure. Twenty-two pigs were studied after 16 weeks of ARAS, ARAS treated with a SW regimen (bi-weekly for 3 weeks) with or without subsequent intrarenal delivery of adipose tissue-derived MSCs and controls. Four weeks after treatment, single-kidney renal blood flow (RBF) before and after infusion of acetylcholine, glomerular filtration rate (GFR), and oxygenation were assessed in vivo and the renal microcirculation, fibrosis, and oxidative stress ex vivo. Mean arterial pressure remained higher in ARAS, ARAS + SW, and ARAS + SW + MSC compared with normal. Both SW and SW + MSC similarly elevated the decreased stenotic kidney GFR and RBF observed in ARAS to normal levels. Yet, SW + MSC significantly improved RBF response to acetylcholine in ARAS, and attenuated capillary loss and oxidative stress more than SW alone. Density of larger microvessels was similarly increased by both interventions. Therefore, although significant changes in functional outcomes were not observed in a short period of time, adjunct MSCs enhanced pro-angiogenic effect of SW to improve renal microvascular outcomes, suggesting this as an effective stratege for long-term management of renovascular disease.

Keywords: extracorporeal shockwave; progenitor cells; renal artery stenosis; renovascular endothelial function.

Figure 1.

A. Schematic of a 16-wk experimental protocol, with 6 shockwave (SW) therapy sessions delivered over a 3-week regimen (weeks 9, 10, and 11; each session indicated by a red arrow), and autologous adipose tissue-derived mesenchymal stem/stromal cells (MSCs) collection at the beginning of week 9 and injection at the end of week 11 (black arrow). B. Characterization of MSC by markers (CD105 and CD90 by Flow cytometry) and proliferation rate in vitro. Single-color controls were acquired to make a compensation matrix for each test, and the signal visually gauged when gating for positive cells. C. CellTrace™ Far Red-labeled MSCs (white arrow) were detected only in stenotic kidneys injected with MSC (top, x10 images), but not in negative control untreated with MSCs. Cytokeratin (green) was stained as reference for tubular cells.

Figure 2.

A-C: Single-kidney glomerular filtration rate (GFR) and renal blood flow (RBF) in pigs with atherosclerotic renal artery stenosis (ARAS), untreated or treated with a shockwave (SW) regimen with or without an intra-renal delivery of adipose tissue-derived mesenchymal stem/stromal cells (MSCs). RBF and GFR of the stenotic ARAS kidney decreased compared to normal, but increased by both SW and SW+MSC to a similar extent. Compared to Normal, the ARAS and ARAS+SW groups had blunted RBF responses to acetylcholine (Ach), which was normalized in the ARAS+SW+MSC group. D: Representative images of blood-oxygen-level-dependent magnetic resonance imaging and micro-computed tomography, and quantification of microvascular density and hypoxia (R2*). E: Cortical and medullary oxygenation was decreased in ARAS compared to Normal, and both were improved by SW and SW+MSC. F: Both SW and SW+MSC improved cortical vascular density that was decreased in ARAS. *p<0.05 vs. Normal, † p<0.05 vs. ARAS (n=5-6/group). Results are mean±SEM.

Figure 3.

A: Representative H&E-stained cortical and medullary sections (x40 images) and immunofluorescent staining of von Willebrand factor (vWF) (x10, positive staining red, nuclei blue). Capillaries (yellow arrow) were identified by the presence of lumen, red blood cells, and/or endothelial cell lining. B: The number of capillaries-per-tubule decreased in ARAS compared to Normal (H&E, performed at x100), but increased after SW and SW+MSC in ARAS. Cortical capillary density in ARAS+SW+MSC was higher than in ARAS+SW. vWF expression in cortex and medulla was diminished in ARAS and ARAS+SW, but restored to normal in ARAS+SW+MSC. *p<0.05 vs. Normal, †p<0.05 vs. ARAS, ‡p<0.05 vs. ARAS+SW (n=5-6/group). Results are mean±SEM.

Figure 4.

A: Representative images of dihydroethidium (DHE) (40×, positive staining pink, nuclei blue], immunofluorescence staining for expression of 8-hydroxy-2' -deoxyguanosine (8-OHdG) (10×, green, nuclei blue) and oxidized low density lipoprotein (Ox-LDL) (10×, red, nuclei blue). B: Renal oxidative stress evaluated by DHE in ARAS+SW was not different from ARAS, whereas SW+MSC decreased renal oxidative stress in ARAS. Increased expression of 8-OHdG in ARAS was attenuated by SW, and further by SW+MSC. SW and SW+MSC similarly decreased elevated expression of Ox-LDL in ARAS. *p<0.05 vs. Normal, † p<0.05 vs. ARAS (n=5-6/group). *p<0.05 vs. Normal, † p<0.05 vs. ARAS, ‡p<0.05 vs. ARAS+SW (n=5/6). Results are mean±SEM.

Figure 5.

A: Renal expression of endothelial nitric oxide synthase (eNOS) and monocyte chemoattractant protein (MCP)-1. B: Expression of eNOS increased in ARAS+SW+MSC compared to all other groups; MCP-1 expression increased in ARAS and was normalized by all treatments. C: Representative trichrome-stained cortex and medulla (x20). D: ARAS increased trichrome staining compared to Normal, which was alleviated both by SW and SW+MSC. *p<0.05 vs. Normal, †p<0.05 vs. ARAS, ‡p<0.05 vs. ARAS+SW(n=5-6/group). Results are mean±SEM.