Adipose tissue-derived mesenchymal stem cells improve revascularization outcomes to restore renal function in swine atherosclerotic renal artery stenosis

Abstract

Background

Reno-protective strategies are needed to improve renal outcomes in patients with atherosclerotic renal artery stenosis (ARAS). Adipose tissue-derived mesenchymal stem cells (MSCs) can promote renal regeneration, but their potential for attenuating cellular injury and restoring kidney repair in ARAS has not been explored. We hypothesized that replenishment of MSC as an adjunct to percutaneous transluminal renal angioplasty (PTRA) would restore renal cellular integrity and improve renal function in ARAS pigs.

Methods and Results

Four groups of pigs (n=7 each) were studied after 16 weeks of ARAS, ARAS 4 weeks after PTRA and stenting with or without adjunct intra-renal delivery of MSC (10×10⁶ cells), and controls. Stenotic kidney blood flow (renal blood flow[RBF]) and glomerular filtration rate (GFR) were measured using multidetector computer tomography (CT). Renal microvascular architecture (micro-CT), fibrosis, inflammation, and oxidative stress were evaluated ex-vivo.

Four weeks after successful PTRA, mean arterial pressure fell to a similar level in all revascularized groups. Stenotic kidney GFR and RBF remained decreased in ARAS (p=0.01 and p=0.02) and ARAS+PTRA (p=0.02 and p=0.03) compared to normal, but rose to normal levels in ARAS+PTRA+MSC (p=0.34 and p=0.46 vs. normal). Interstitial fibrosis, inflammation, microvascular rarefaction, and oxidative stress were attenuated only in PTRA+MSC-treated pigs.

Conclusions

A single intra-renal delivery of MSC in conjunction with renal revascularization restored renal hemodynamics and function, and decreased inflammation, apoptosis, oxidative stress, microvascular loss, and fibrosis. This study suggests a unique and novel therapeutic potential for MSC in restoring renal function when combined with PTRA in chronic experimental renovascular disease.

INTRODUCTION

Renal artery stenosis (RAS) is one of the reversible mechanisms for hypertension. Atherosclerosis is the most common cause of RAS, accounting for 90% of the cases¹. Based upon community-based screening, atherosclerotic RAS (ARAS) exceeding 60% lumen occlusion averages 6.8% in the elderly population². ARAS can accelerate hypertension and lead to loss of kidney function, which are known to increase cardiovascular morbidity and mortality³.

Renal revascularization using endovascular percutaneous transluminal renal angioplasty (PTRA) and stenting has been a common treatment strategy in patients with ARAS both to reduce blood pressure and improve renal function. To date, however, randomized, prospective trials fail to identify major benefits from restoring blood flow for preservation of renal function^{4, 5} compared to medical therapy alone. This might be due to lingering kidney tissue damage that is not reversed by restoring blood flow with PTRA alone. In line with these clinical observations, we have previously shown in a swine model of non-atherosclerotic RAS that PTRA partially restores the renal microvascular network and improves renal function, but vascular wall remodeling and fibrosis are incompletely reversed⁶.

The presence of an atherosclerotic environment compounds these effects. Renal revascularization in a swine model of ARAS normalize blood pressure levels, but fails to improve tubulointerstitial injury, microvascular rarefaction, and renal function in the stenotic kidney⁷. This dissociation between the effects of revascularization on blood pressure and renal function underscores the need to identify more effective strategies to restore the structures with the stenotic kidney in ARAS in addition to PTRA.

Our previous studies demonstrated that intra-renal delivery of autologous hematopoietic endothelial progenitor cells (EPC) can increase neovascularization and mitigate renal injury in non-atherosclerotic RAS⁸. However, the capacity of this cell-based therapy to reverse the more profound damage observed in the ARAS kidney was more limited in that EPC only partially improved microvascular density and failed to fully restore renal blood flow (RBF) and glomerular filtration rate (GFR)⁹. We speculated that both securing renal arterial patency, and at the same time improving the regenerative capacity of the post-stenotic kidney using cell-based therapy, might be a more effective strategy to preserve the stenotic kidney. As a practical matter, autologous EPC are difficult to isolate and expand. Mesenchymal stem cells (MSC) are undifferentiated non-embryonic stem cells present in adult tissues, which have the ability to differentiate into a broad spectrum of cell lineages¹⁰. Moreover, MSC can be isolated from a variety of tissues, including adipose tissue and bone marrow, and possess immunomodulatory properties that decrease inflammation and immune responses¹¹.

Previous studies showed that MSC restore renal structure and function in experimental rodent models of acute renal failure¹². Whether MSC might augment renal function and structure improvement in response to PTRA in a large animal model remains unknown. Thus, we hypothesized that intra-renal infusion of allogeneic MSC at the time of revascularization would restore renal cellular integrity and repair mechanisms in experimental ARAS.

RESULTS

Six weeks after induction of RAS and before PTRA, all ARAS pigs demonstrated hemodynamically significant stenosis (79.4±2.7%, p=0.26 ANOVA)¹³, and mean arterial pressure (MAP) was elevated compared to normal pigs (p<0.01 in all).

The systemic characteristics in all pigs 4 weeks after PTRA or sham are summarized in Table 1. Total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were elevated in all ARAS groups compared to normal. As common in chronic ARAS^{14, 15}, plasma renin activity (PRA) levels were similar among the groups.

Table 1.

Systemic characteristics (mean±SEM) in normal, ARAS, ARAS+PTRA, and ARAS+PTRA+MSC pigs (n=7 each) 4 weeks after PTRA or sham.

	NORMAL	ARAS	ARAS+PTRA	ARAS+PTRA+MSC
Body weight (Kg)	54.0±6.2	55.1±4.3	55.9±7.1	55.8±5.6
Degree of stenosis (%)	0	87.2±21.1*†	0	0
Mean arterial pressure (mmHg)	97.3±11.9	145.3±19.6*†	102.7±5.9	97.8±4.8
Serum creatinine (mg/dL)	1.30±0.14	1.83±0.33*†	1.89±0.31*†	1.45±0.14
PRA (ng/ml/hr)	0.14±0.09	0.13±0.12	0.19±0.08	0.15±0.08
Total cholesterol (mg/dL)	92.5±16.0	481.0±80.9*	415.7±126.6*	403.4±99.2*
Triglycerides (mg/dL)	7.8±1.9	9.1±2.3	8.2±4.8	6.6±1.7
HDL cholesterol (mg/dL)	43.7±12.8	159.3±57.9*	148.2±62.0*	137.9±48.4*
LDL cholesterol (mg/dL)	47.3±8.6	302.6±81.4*	265.9±101.6*	291.6±85.9*
8-isoprostane (pg/ml)	103.2±9.9	195.3±0.8*†	187.0±47.9*†	124.0±11.95
Interleukin 1β (pg/ml)	28.6 ± 14.5	373.3 ± 145.7*†	234.6 ± 98.3*†	71.5 ± 41.5
Urinary albumin (ug/mL)	3.65 ± 1.07	3.80 ± 0.67	3.14 ± 1.46	3.22 ± 0.33

^*p≤0.05 vs. normal.

^†p<0.05 vs. ARAS+PTRA+MSC.

PRA: plasma renin activity; HDL: high-density lipoprotein; LDL: low-density lipoprotein.

PTRA successfully reduced blood pressure

There was no residual stenosis at 16 weeks in PTRA-treated pigs (Figure 1A). Continuously measured mean arterial pressure (MAP) decreased immediately after PTRA and persisted at normal levels until the end of the study (p<0.05 vs. ARAS, p>0.05 vs. Normal) (Table 1, Figure 1B).

Figure 1.

A: Renal angiography in a pig with atherosclerotic renal artery stenosis (ARAS) before (left) and 4 weeks after (right) revascularization with percutaneous transluminal renal angioplasty (PTRA). B: Mean arterial pressure measured using telemetry decreased after PTRA. C: Single-kidney RBF and GFR were restored in MSC-treated pigs, although RBF response to Ach remained blunted. *p<0.05 vs. normal, †p<0.05 vs. ARAS+PTRA+MSC, ‡p<0.05 vs. baseline.

MSC characterization and culture

MSC displayed a fibroblast-like, spindle-shaped morphology (Figure 1As), expressed CD44 CD90, and CD105 markers (Figure 2As), and secreted vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-α in the culture media (Figure 1Cs). Furthermore, MSC successfully transdifferentiated into osteocytes, chondrocytes, and adipocytes in vitro, supporting their mesenchymal origin (Figure 1Bs).

Figure 2.

A: Micro-CT 3D images of the kidney showing improved microvascular architecture in ARAS+PTRA+MSC. Transmural spatial density (B) and its classification by vessel size (C), average vessel diameter (D), and tortuosity (E) of renal cortical microvessels. F: Renal protein expression of vascular endothelial growth factor (VEGF) was downregulated in ARAS and ARAS+PTRA, but improved in ARAS+PTRA+MSC. *p<0.05 vs. normal, †p<0.05 vs. ARAS+PTRA+MSC.

MSC home to the stenotic kidney

The MSC retention rate 4 weeks after intra-arterial administration was 13.1±2.2%. CM-DiI-labeled MSC were mostly detected at the renal cortical interstitium 4 weeks after injection (Figure 2Bs).

Histological analysis showed no evidence of cellular rejection (e.g. CD3 clusters), micro-infarcts, or tumors in tissue sections from ARAS+PTRA+MSC pigs.

MSC restore renal hemodynamics and function

Basal stenotic kidney GFR and RBF were similarly attenuated in ARAS and ARAS+PTRA (Figure 1C, p<0.05 vs. normal), but restored to normal levels in ARAS+PTRA+MSC. GFR responses to acetylcholine (Ach) were normalized in MSC-treated pigs, while RBF responses remained blunted (p=0.32 vs. baseline). Serum creatinine levels were higher in ARAS compared to normal, and remained elevated after PTRA (p=0.04 vs. normal; p=0.77 vs. ARAS). Treatment with MSC led to a fall in serum creatinine to normal levels (p=0.14 vs. normal, Table 1).

Microvascular architecture is improved in MSC-treated pigs

Transmural spatial density of cortical microvessels was similarly diminished in ARAS and ARAS+PTRA, but improved after MSC treatment to levels not different from normal pigs (Figure 2A–B). In particular, the number of small vessels (<40um) was significantly reduced in ARAS and ARAS+PTRA compared to normal, but normalized after MSC administration (Figure 2C, p=0.27 vs. normal). Vessel diameter was similarly increased in ARAS and ARAS+PTRA compared to normal, but decreased to normal levels in MSC-treated pigs (Figure 2D, p=0.12 vs. normal). Tortuosity (a measure of angiogenesis) was increased in MSC-treated pigs compared to normal, but was lower than in ARAS (p=0.03) and ARAS+PTRA (Figure 2E, p=0.05) pigs. In addition, expression of VEGF was reduced in ARAS and ARAS+PTRA (Figure 2F, p<0.05 vs. normal), but restored to normal levels in MSC-treated pigs (p<0.05 vs. ARAS and ARAS+PTRA, p=0.61 vs. normal).

MSC reduced oxidative stress

Circulating levels of 8-isoprostanes were significantly higher in sham-treated and PTRA-treated ARAS compared to normal (p=0.04 and p=0.03, respectively), but were restored to normal levels after renal administration of MSC (Table 1, p>0.05 vs. normal). Moreover, in-situ production of superoxide anion was similarly increased in ARAS and ARAS+PTRA (p=0.01 vs. normal, p=0.99 vs. ARAS) and decreased to levels not different from normal in ARAS+PTRA+MSC (Figure 3A, p=0.10 vs. normal). Also, the increased protein expression of the NAD(P)H-oxidase subunit p47phox observed in ARAS and ARAS+PTRA was normalized after MSC treatment, suggesting a decreased potential for superoxide generation. Furthermore, protein expression of nitrotyrosine (NT), which was similarly and significantly elevated in ARAS and ARAS+PTRA kidneys compared with normal (p<0.05), was substantially reduced in MSC-treated pigs (Figure 3B, p<0.05 vs. ARAS and ARAS+PTRA, p=0.29 vs. Normal), implying decreased production of peroxynitrite.

Figure 3.

Oxidative stress declined after PTRA+MSC. A: Renal production of superoxide anion (top), detected by dihydroethidium (40×), and its quantification (bottom). B: Representative immunoblots and renal protein expression of nitrotyrosine (NT) and p47 in Normal, ARAS, ARAS+PTRA, and ARAS+PTRA+MSC. *p<0.05 vs. normal, †p<0.05 vs. ARAS+PTRA+MSC.

MSC decreased inflammation in the stenotic kidney

The numbers of CD45+, CD3+ and CD163+ cells infiltrating the kidney were similarly increased in ARAS and ARAS+PTRA pigs compared to normal (p<0.05 for both), but decreased to normal levels after treatment with MSC (Figure 4A–D). Although MSC failed to fully restore renal expression of TNF-α (p=0.03 vs. normal, Figure 4E), it tended to decrease compared to ARAS and ARAS+PTRA pigs (p=0.08 and p=0.09, respectively). Moreover, systemic levels of interleukin (IL)-1β, which were significantly increased in ARAS (p=0.02 vs. normal) and ARAS+PTRA (p=0.04) pigs, were restored to normal levels in animals treated with MSC (p=0.1 vs. normal) (Table 1).

Figure 4.

PTRA+MSC decreased renal inflammation. Representative immunostaining (40×) of B-T lymphocytes (CD45+), T-lymphocytes (CD3+), and macrophages (CD163+) (A) and their quantification (B, C, and D). Renal protein expression of TNF-α increased in ARAS and ARAS+PTRA, and tended to decline in ARAS+PTRA+MSC, although it remained higher than normal (E). *p<0.05 vs. normal, †p<0.05 vs. ARAS+PTRA+MSC.

Renal scarring was reduced in ARAS+PTRA+MSC

Tubulo-interstitial fibrosis observed in ARAS and ARAS+PTRA pigs was attenuated in ARAS+PTRA+MSC (Figure 5A–B, p=0.02 vs. ARAS, p=0.05 vs. ARAS+PTRA, p=0.12 vs. normal). Furthermore, glomerular score, which did not improve after revascularization alone, declined in MSC-treated pigs (Figure 5C, p=0.002 vs. ARAS, p<0.04 vs. ARAS+PTRA, p=0.29 vs. normal). Collagen content, assessed by Sirius red, was similarly elevated in ARAS and ARAS+PTRA compared to normal (p<0.05 for both), but decreased in PTRA+MSC-treated animals (Figure 6A, p=0.05 vs. ARAS, and ARAS+PTRA, p=0.07 vs. normal). PTRA alone also failed to restore the expression of the fibrogenic factor transforming growth factor (TGF)-β1 (Figure 6B, p<0.05 vs. normal), but its levels were significantly reduced in ARAS+PTRA+MSC (p=0.04 vs. ARAS, p=0.04 vs. ARAS+PTRA, p=0.73 vs. normal).

Figure 5.

A: Representative renal trichrome staining (40×) in normal, ARAS, ARAS+PTRA and ARAS+PTRA+MSC pigs. Glomerular and tubulointerstitial fibrosis (B) and glomerular score (C, % of sclerotic glomeruli) decreased after PTRA+MSC. *p<0.05 vs. normal, †p<0.05 vs. ARAS+PTRA+MSC.

Figure 6.

A: Representative Sirius red staining, under polarized light (top) and its quantification (bottom). B: Representative immunoblots and densitometric quantification of TGF-β1. *p<0.05 vs. normal, †p<0.05 vs. ARAS+PTRA+MSC.

No significant differences were found in the expression of podocin and nephrin among the groups (data not shown) or urinary albumin excretion (Table 1).

DISCUSSION

This is, to the best of our knowledge, the first study demonstrating that adjunctive cell-based therapy in conjuction with restoration of vascular patency can improve renal structural and functional responses in experimental ARAS. Intra-renal administration of adipose-tissue derived MSC improved renal function and structure 4 weeks after revascularization and reduced oxidative stress, fibrosis, inflammation, and microvascular remodeling in the stenotic ARAS kidney. This may thus provide a novel therapeutic approach to preserve the stenotic kidney.

Although renal stenting is commonly employed in clinical ARAS, data supporting PTRA and stenting versus medical therapy are controversial¹⁶. Restoring vessel patency alone has been shown to confer limited benefit as regarding recovery of kidney function^{17, 18}. Consequently, the use of this procedure has declined considerably over the past few years. One result of this trend has been the alarming increase in the appearance of medically treated subjects with more severe kidney injury beyond the stenotic lesion.

We have recently shown in a swine model of non-atherosclerotic that PTRA succesfully restored blood pressure, GFR, and renal endothelial function, but not RBF or fibrosis in the stenotic kidney⁶. We have also shown in a swine model of ARAS that PTRA improved blood pressure, but failed to restore renal function beyond the stenotic lession⁷. The present study extends these observations and undescores the ability of PTRA to reverse renovascular hypertension, but not necessarily dysfunction in the stenotic ARAS kidney. Thus, whereas blood pressure may be controlled succesfully with antihypertensive medication and/or revascularization, renal dysfunction requires additional strategies, targeted to repair the kidney parenchyma.

Several previous studies demonstrated that MSC can protect the kidney from ischemia/reperfusion injury and stimulate renal parenchymal regeneration^{12, 19–22}. Ninichuk and colleagues also demonstrated that weekly injections with MSC prevent loss of peritubular capillaries and reduce interstitial fibrosis in collagen4a3-deficient mice²³. Our study now shows in a large animal model that intra-arterially delivered MSC restored basal hemodynamics and function in the kidneys beyond a stenotic lesion. These observations were accompained by normalized GFR (although not RBF) responses to endothelium-dependent chalenge with Ach, suggesting improvement of endothelial function.

We have previously shown that impaired renal function in ARAS is associated with oxidative stress and decreased expression of angiogenic factors, leading to microvascular loss and renal dysfunction in the stenotic kidney²⁴. In the present study, both renal oxidative stress, assessed by the in situ production of superoxide anion, and systemic oxidative stress as reflected by isoprostane levels, were decreased in MSC-treated pigs. This anti-oxidant effect was also illustrated by the decreased expression of NAD(P)H-oxidase (p47phox) and peroxynitrite (nitrotyrosine) formation observed in ARAS+PTRA+MSC pigs. These findings extend previous studies showing an anti-oxidant effect of MSC through modulation of pathways associated with the activation of anti-oxidant pathways such as superoxide dismutase and glutathione peroxidase^{25, 26}.

Because reactive oxygen species in high concentrations inhibit angiogenesis, the decreased oxidative stress in PTRA+MSC-treated pigs may have contributed to the improved microvascular architecture reflected by their increased spatial density. Specifically, a selective loss of small microvessels, which led to increased average vessel diameter in ARAS and ARAS+PTRA, was substantially improved in ARAS+PTRA+MSC pigs. Alternatively, the improved microcirculation might have resulted from increased renal expression of the pro-angiogenic factor VEGF, possibly via paracrine production or an indirect effect of MSC. Previous studies in an acutely ischemic rat model suggested that VEGF is an important mediator of the renoprotective effect of MSC²⁷. Likewise, the potent angiogenic properties of MSC were supported in our study by the active secretion of the pro-angiogenic factor VEGF in cell culture. Despite these effects, the blunted RBF reactivity we observed suggested residual endothelial dysfunction and that restoration of the microcirculation was incomplete.

The current study also showed that treatment with MSC reduced renal inflammation, as evidenced by decreased CD163+ macrophage infiltration as well as CD45+ and CD3+ lymphocytes in the stenotic kidneys of the ARAS+PTRA+MSC animals. In addition, IL1-β levels and renal expression of TNF-α decreased in MSC-treated pigs. This anti-inflammatory effect of MSC might be related to their immunomodulatory properties, as well as paracrine release of anti-inflammatory cytokines, which may influence surrounding parenchymal cells in the stenotic kidney. Indeed, MSC have the ability to inhibit maturation of dendritic cells, and suppress the function of B cells, T cell, and natural killer cells, by decreasing the surface expression of class I and II major histocompatibility molecules and the expression of costimulatory molecules²⁸. Notably, our observations contrast with recent studies in a porcine model of ischemia reperfusion injury that reported limited-immune modulating activity of MSC with no beneficial for kidney function and histology²⁹. However, the different study design (bilateral acute kidney injury model) and the fact that bone marrow-derived MSC were infused into the suprarenal aorta (not intra-renal) might partly explain the different results obtained in our study.

The protective effect of MSC in the stenotic ARAS kidney might be attributed partly to their capacity to engraft in the damaged kidney. Four weeks after their infusion, MSC were detected in renal tissue sections, mostly at the insterstitium. This observation may partly explain their ability to decrease inflammation, as T cells and macrophages tend to infiltrate the interstitium³⁰. Similarly, by decreasing rarefaction of interstitial vessels, which is an important determinant of GFR³¹, MSC might have restored renal function beyond the stenotic lesion. The recruitment of exogenous MSC to the injured renal tissue may be mediated by their expression of the multifunctional receptor CD44, as shown in mice models of acute renal failure ³². In addition, we have shown that ARAS kidney releases a plethora of injury signals and homing factors that attract circulating progenitor cells⁹, which likely also promote adhesion and retention of exogenously-delivered MSC.

Although ARAS can sometimes lead to proteinuria³³, no differences in urinary protein excretion were found among the groups, and renal expressions of podocin and nephrin were similar, arguing against structural alterations in the glomerular basement membrane in this early stage of the disease. On the other hand, delivery of MSC in conjunction with PTRA decreased the number of sclerotic glomeruli in the stenotic kidney. Downregulation of the fibrogenic factor TGF-β1 might have contributed to the reduction in tubulointerstitial fibrosis and glomerulosclerosis in the stenotic kidney of MSC-treated pigs. Our results are underscored by previous observations showing a renoprotective effect of arterially delivered MSC decreasing renal fibrosis in rats with unilateral ureteral obstruction³⁴.

Limitations

Our study is limited by the short duration of ARAS, lack of additional comorbid conditions, and use of relatively young animals. However, similar to observations in humans, PTRA alone failed to improve within 4 weeks renal functional compromise, intra-renal inflammation and fibrosis in the RAS kidney distal to the stenosis. While blood pressure does not consistently decline in patients after revascularization as it did in our experimental model, human ARAS might be superimposed on essential hypertension or pre-existing kidney disease. In addition, a potential bias in the calculation of MSC retention rate is the fact that their in vitro migration capabilities are largely influenced by the systemic and local inflammatory state ³⁵. Future studies are needed to examine the persistence of the beneficial effects of MSC on the response to PTRA over longer periods of time and in humans.

Conclusions

Taken together, our observations have identified a synergistic beneficial role of adipose-tissue-derived MSC and PTRA in reversing renal injury induced by ARAS. The beneficial effect of MSC delivered after PTRA appears to be mediated by improvement of microvascular remodeling and reduction of oxidative stress and inflammation in the stenotic kidney via paracrine mechanisms. Hence, our data indicate that intra-arterial administration of MSC constitutes an effective approach to improve the response to revascularization in swine ARAS. Additional studies are needed to provide further insight into the role of MSC in improving renal function and response to revascularization in this increasingly prevalent disease.

CONCISE METHODS

Twenty-eight domestic female pigs (50–60kg) were studied during 16 weeks of observation (Figure 7) after approval by the Institutional Animal Care and Use Committee.

At baseline, pigs were randomized into 2 groups, which were fed either a 2% high-cholesterol diet (n=21), as a surrogate for early atherosclerosis, or normal pig chow (n=7). Six weeks later, all animals were anesthetized with 0.5g of intramuscular ketamine and xylazine, and anesthesia then maintained with intravenous ketamine (0.2mg/kg/min) and xylazine (0.03mg/kg/min). RAS was induced in high-cholesterol animals by placing a local-irritant coil in the main renal artery, leading to a gradual development of unilateral RAS, as previously described³⁶. The rationale for initiating diet before RAS was to reproduce the clinical situation in ARAS in which early atherosclerosis precedes the stenosis³⁷. A sham procedure, which involved cannulating the renal artery (without placement of irritant coil), was performed in normal animals. In addition, a telemetry system was implanted in the left femoral artery to continuously measure MAP for 10 additional weeks.

Six weeks after induction of RAS (Figure 1), animals were similarly anesthetized and the degree of stenosis determined by angiography. Fourteen ARAS pigs were treated with PTRA while the others underwent a sham procedure. Immediately after PTRA, labeled allogeneic adipose tissue-derived MSC isolated from normal swine were delivered into the stenotic kidney of 7 ARAS+PTRA pigs. A vehicle (saline) was administered in other 7 ARAS+PTRA pigs.

Four weeks after PTRA or sham, the pigs were again similarly anesthetized and the degree of stenosis determined by angiography. Blood samples were collected from the inferior vena cava for PRA, total cholesterol, triglycerides, HDL, LDL, IL-1β, and creatinine measurements. In addition, urine samples were collected and albumin concentration quantified by ELISA (Bethyl Laboratories, Texas). Renal hemodynamics and function in each kidney were then assessed using multi-detector computer tomography (MDCT).

Two days after completion of all studies, pigs were euthanized with a lethal intravenous dose of 100mg/kg of sodium pentobarbital (Sleepaway, Fort Dodge Laboratories, Inc., Fort Dodge, Iowa)³⁸. The kidneys were removed using a retroperitoneal incision and immediately dissected, and sections were frozen in liquid nitrogen (and maintained at −80°C) or preserved in formalin¹⁵ for in vitro studies.

In-vivo studies

Renal hemodynamics and function

Basal regional perfusion, RBF, and GFR in each kidney were noninvasively assessed using MDCT, an ultra-fast scanner that provides accurate and noninvasive quantifications of single kidney volume, hemodynamics, and function^{14, 15, 39}. In brief, 160 consecutive scans were performed following a central venous injection of iopamidol (0.5mL/kg per 2 seconds). The same procedure was repeated after 15 min toward the end of a 10 min suprarenal infusion of acetylcholine (Ach, 5 mg/kg/min) to test endothelium-dependent microvascular reactivity¹⁴. Images were reconstructed and displayed with the Analyze™ software package (Biomedical Imaging Resource, Mayo Clinic, Rochester, MN). Tissue attenuation curves obtained from cross-sectional images from the aorta, renal cortex, and medulla were fitted by curve-fitting algorithms to obtain measures of renal function^{6, 7, 14}. Cortical and medullary volumes were calculated with planimetry.

In vitro studies

Renal morphology and fibrosis

Midhilar 5-μm cross-sections of each kidney (one per animal) were stained with Masson’s trichrome and examined using an image-analysis program (MetaMorph®, Meta Imaging Series 6.3.2, Allentown, PA). In each slide, staining intensity was semiautomatically quantified in 15–20 fields, expressed as fraction of kidney surface area, and the results from all fields were averaged⁴⁰. Glomerular score (% of sclerotic out of 100 glomeruli) was also assessed^{14, 15}. Glomerular and tubulointerstitial collagen content was evaluated by Sirius red staining as previously described ⁴¹. Slides were visualized under polarized light microscope, and pictures of the entire slice were taken with identical exposure settings for all sections and analyzed using MetaMorph®. Results were quantified as percent area staining. In addition, western blotting protocols were followed in each kidney sample using specific polyclonal antibodies against TGF-β1 (Santa Cruz 1:200)¹⁴. All quantifications were performed in a blinded manner.

Oxidative stress

Systemic levels of isoprostanes were assessed using an EIA kit⁴². Renal redox status was evaluated by the in-situ production of superoxide anion, detected by fluorescence microscopy using dihydroethidium (DHE)⁴⁰ and by the expression of the NADPH-oxidase sub-unit p47 (Santa Cruz 1:200), and NT (Cayman Chemical 1:200) determined by Western blotting^{9, 42}.

Inflammation

Renal inflammation was evaluated by standard immunostaining with antibodies against macrophages (anti-macrophage CD163), B-T lymphocytes (CD45), and CD3 T-lymphocytes. Positive cells were manually counted under X60 in random glomerular or cortical fields and averaged from 20 fields in each sample in a blinded manner. In addition, renal expression of TNF-α (Santa Cruz 1:200) was quantified by Western Blot^{37, 43}.

Systemic inflammation was assessed by IL-1β levels, quantified by ELISA for porcine IL-1β developed using Nunc MaxSorp plate (ThermoFisher Scientific, 437796) coated with 100μl of IL-1β capture antibody 2.0 μg/ml (R&D systems, DY681) and 100μl of pig plasma samples. The plate was read by the SynergyMx plate reader (BioTek), set at standard luminescence reading mode, for 1 hour at 10-min intervals. The maximum readout was then further analyzed.

Glomerular damage

The degree of glomerulosclerosis was assessed by glomerular score and podocyte injury by renal expression of the podocyte proteins podocin and nephrin (Abcam 1:200).

Micro-vascular architecture

Stenotic kidneys were perfused with microfil MV122 (an intravascular contrast agent) under physiological pressure using a saline-filled cannula ligated in a segmental artery. Samples were prepared and scanned, and images analyzed as previously described^{6, 41}. Spatial density, average diameter, and tortuosity of renal cortical microvessels (diameters of 20–500μm) were calculated⁴¹ using Analyze™. In addition, renal expression of VEGF (Santa Cruz 1:200) was quantified by Western Blot.

Statistical methods

Statistical analysis was performed using JMP software package version 8.0 (SAS Institute Inc. Cary, NC). The Shapiro-Wilk test was used to test for any deviation from normality. Results were expressed as mean±SEM for normally distributed variables. Comparisons within groups were performed using the paired Student t-test and among groups using ANOVA followed by the unpaired t-test with Bonferroni correction. Data that did not show a Gaussian distribution were expressed as median (range) and comparisons within and among the groups performed using non-parametric tests (Wilcoxon and Kruskal Wallis, respectively). Statistical significance for all tests was accepted for p≤0.05.