Obesity-metabolic derangement preserves hemodynamics but ...

Am J Physiol Renal Physiol 305: F265–F276, 2013. First published May 8, 2013; doi:10.1152/ajprenal.00043.2013.

Obesity-metabolic derangement preserves hemodynamics but promotes intrarenal adiposity and macrophage infiltration in swine renovascular disease Xin Zhang,1 Zi-Lun Li,1,2 John R. Woollard,1 Alfonso Eirin,1 Behzad Ebrahimi,1 John A. Crane,1 Xiang-Yang Zhu,1 Aditya S. Pawar,1 James D. Krier,1 Kyra L. Jordan,1 Hui Tang,1 Stephen C. Textor,1 Amir Lerman,2 and Lilach O. Lerman1,3 1

Division of Nephrology and Hypertension, Mayo Clinic, Rochester, Minnesota; 2Division of Vascular Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guang Zhou, China; and 3Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota Submitted 23 January 2013; accepted in final form 27 April 2013

Zhang X, Li ZL, Woollard JR, Eirin A, Ebrahimi B, Crane JA, Zhu XY, Pawar AS, Krier JD, Jordan KL, Tang H, Textor SC, Lerman A, Lerman LO. Obesity-metabolic derangement preserves hemodynamics but promotes intrarenal adiposity and macrophage infiltration in swine renovascular disease. Am J Physiol Renal Physiol 305: F265–F276, 2013. First published May 8, 2013; doi:10.1152/ajprenal.00043.2013.—Obesity-metabolic disorders (ObM) often accompany renal artery stenosis (RAS). We hypothesized that the coexistence of ObM and RAS magnifies inflammation and microvascular remodeling in the stenotic kidney (STK) and aggravates renal scarring. Twenty-eight obesity-prone Ossabaw pigs were studied after 16 wk of a high-fat/high-fructose diet or standard chow including ObM-sham, ObM-RAS, Lean-RAS, or Leansham (normal control) groups. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed by multidetector computed tomography (CT), renal oxygenation and tubular transport capability by blood-oxygen-level-dependent MRI, and microcirculation by micro-CT for vessel density, and Western blotting for protein expressions of angiogenic factors (VEGF/FLK-1). Renal vein and inferior vena cava levels of inflammatory cytokines were measured to evaluate systemic and kidney inflammation. Macrophage (MØ) infiltration and subpopulations, fat deposition in the kidney, and inflammation in perirenal and abdominal fat were also examined. GFR and RBF were decreased in Lean-STK but relatively preserved in ObMSTK. However, ObM-STK showed impaired tubular transport function, suppressed microcirculation, and stimulated glomerulosclerosis. ObM diet interacted with RAS to blunt angiogenesis in the STK, facilitated the release of inflammatory cytokines, and led to greater oxidative stress than Lean-STK. The ObM diet also induced fat deposition in the kidney and infiltration of proinflammatory M1-MØ, as also in perirenal and abdominal fat. Coexistence of ObM and RAS amplifies renal inflammation, aggravates microvascular remodeling, and accelerates glomerulosclerosis. Increased adiposity and MØ-accentuated inflammation induced by an ObM diet may contribute to structural injury in the post-STK kidney. renal artery stenosis; obesity-metabolic derangement; macrophage; inflammation; adiposity

is on the rise world-wide (14, 27, 28). Obesity is associated with hypertension, diabetes, and atherosclerosis (37), and contributes greatly to cardiovascular morbidity and mortality. Obesity has also been identified to initiate chronic kidney disease, affect the progression of preexisting renal diseases (3, 15), and is a strong independent risk factor for end-stage renal disease (16).

THE PREVALENCE OF OBESITY

Address for reprint requests and other correspondence: L. O. Lerman, Div. of Nephrology and Hypertension, Mayo Clinic, 200 First St. SW, Rochester, MN (e-mail: [email protected]). http://www.ajprenal.org

Importantly, the constellation of metabolic derangements (ObM) that often accompanies obesity, including insulin resistance, elevated blood pressure, and atherogenic dyslipidemia (e.g., hypertriglyceridemia), contributes to the pathophysiological changes during obesity-related tissue injury, including the kidney (31). Although the underlying mechanism mediating kidney damage in ObM is incompletely understood, adaptations to increased body mass/excretory load or sodium retention, adverse effects of insulin resistance, excess of lipid products in the kidney, inflammation, and activated renin-angiotensin system have been all considered (2). Inflammatory cytokines implicated as important mediators of ObM-related kidney injury include TNF-␣, IFN-␥, IL-6, leptin, and monocyte chemoattractant protein (MCP)-1, which might be derived from the cytokine-rich adipose tissue (31). In addition, the adaptation to increased body mass or excretory load also plays important role in the renal pathology by elevating the renal blood flow (RBF) and increasing the workload of the kidney (31). Renal artery stenosis (RAS) often increases inflammation, microvascular remodeling, and tissue scarring in the kidney due to hypoperfusion and vasoconstriction (21, 38). These deleterious processes might be exacerbated as a result of superimposed co-morbid conditions. However, whether concurrent ObM aggravates poststenotic kidney (STK) injury and the potential pathways have not been elucidated. We hypothesized that coexistence of ObM and RAS amplifies the STK inflammation and structural damage. MATERIALS AND METHODS

Study Protocols This study was approved by the Institutional Animal Care and Use Committee. Twenty-four 3-mo-old littermate Ossabaw pigs (Swine Resource, Indiana University) were randomized in four groups (n ⫽ 7 each) that included RAS and sham pigs fed with an ObM diet (ObM-RAS and ObM-sham; high-fat/high-fructose, 5B4L, Purina Test Diet, Richmond, IN) (22) or standard chow [Lean-RAS and Lean-sham (normal control)]. Diets were fed for a total of 16 wk, with free access to water. Twelve weeks after initiation of diets, RAS induction or sham procedure was performed, followed 4 wk later by determination of renal function and oxygenation using multidetector computed tomography (MDCT) and blood oxygenation level-dependent (BOLD) MRI. Animals were then euthanized, and both kidneys were collected for assessments of the microvasculature, histology, and protein expression. RAS Induction Placement of a local irritant coil in the right main renal artery of the corresponding groups led to a gradual development of unilateral RAS, as previously described (5). Blood pressure was then measured continuously by a telemetry transducer (Data Science International, St. Paul, MN) implanted in the left femoral artery (9, 39).

1931-857X/13 Copyright © 2013 the American Physiological Society

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OBESITY PROMOTES INTRARENAL ADIPOSITY

Table 1. Systemic characteristics in sham and renal artery stenosis (RAS) pigs with or without obesity metabolic derangement (ObM; n ⫽ 7 each) Sham

Body weight, kg Degree of stenosis, % MAP, mmHg Urine mAlb, ␮g/ml Fasting glucose, mg/dl Fasting insulin, ␮U/ml HOMA-IR TC, mg/dl LDL, mg/dl TG, mg/dl Plasma creatinine, mg/dl PRA, ng 䡠 ml⫺1 䡠 h⫺1 Abdominal fat volume fraction, %

RAS

P Value for Two-Way ANOVA

Lean

ObM

Lean

ObM

31.4 ⫾ 4.3

45.0 ⫾ 5.7*

98.9 ⫾ 4.7 6.6 ⫾ 1.4 163.1 ⫾ 25.2 0.45 ⫾ 0.06 3.1 ⫾ 0.4 94.6 ⫾ 2.0 20.1 ⫾ 16.2 21.6 ⫾ 4.2 1.1 ⫾ 0.0 0.02 ⫾ 0.01 27.7 ⫾ 2.8

112.2 ⫾ 7.7 21.5 ⫾ 7.9 179.3 ⫾ 27.4 0.83 ⫾ 0.12* 6.6 ⫾ 0.5* 388 ⫾ 48.1* 242.9 ⫾ 21.9* 47.0 ⫾ 17.1* 1.1 ⫾ 0.1 0.10 ⫾ 0.03 38.2 ⫾ 5.1*

35.7 ⫾ 2.1 87.9 ⫾ 4.9 122.2 ⫾ 4.1* 153.5 ⫾ 70.4* 166.2 ⫾ 31.1 0.35 ⫾ 0.13 1.9 ⫾ 0.5 85.4 ⫾ 5.0 32.4 ⫾ 3.0 18.5 ⫾ 3.2 1.2 ⫾ 0.1 0.07 ⫾ 0.02 30.2 ⫾ 2.1

45.3 ⫾ 1.2* 85.6 ⫾ 4.7 136.2 ⫾ 5.5*† 221.1 ⫾ 96.0*† 184.6 ⫾ 28.6 0.98 ⫾ 0.21* 9.3 ⫾ 3.2* 346.7 ⫾ 40.6* 187.3 ⫾ 27.3* 42.2 ⫾ 5.6* 1.3 ⫾ 0.1 0.16 ⫾ 0.06* 41.5 ⫾ 3.2*

Diet

RAS

DietxRAS

0.001

0.54

0.54

⬍0.001 0.009 0.93 0.93 0.77 0.10 0.09 0.44 0.014 0.07 0.38

0.95 0.67 0.68 0.46 0.34 0.17 0.09 0.69 0.68 0.94 0.91

0.021 0.52 0.10 0.008 0.012 ⬍0.001 ⬍0.001 0.027 0.905 0.005 0.003

Values are means ⫾ SE. MAP, mean arterial pressure; mAlb, microalbumin; HOMA-IR, homeostasis model assessment of insulin resistance; TC, total cholesterol; TG, triglycerides; PRA, plasma renin activity. *P ⬍ 0.05 vs. Lean-sham. †P ⬍ 0.05 vs. ObM-sham.

Sham

A

ObM

Baseline

Lean

Lean-STK

x

x

x

♠ RAS

*

D

# Medullary R2*

0.0

Post-furosemide

30.0

*

15.0

x

x

Pre-furosemide

C

x

ObM

RAS LeanSTK

ObMSTK

0.0

#

#

Sham Lean

Delta R2*

30.0

ObM-STK

x

x

Post-furosemide Cortical R2*

B

RAS

* -2.0

15.0 Lean

ObM

Sham

Lean- ObMSTK STK RAS

Lean

ObM

Sham

LeanSTK

ObMSTK

-4.0

♠ RAS

RAS

Fig. 1. Intrarenal oxygenation and medullary tubular function in stenotic (STK) and sham kidneys with or without obesity-metabolic derangement (ObM; n ⫽ 7 each). A: representative kidney blood oxygen level-dependent (BOLD)-MRI images obtained at baseline and after furosemide. Dashed lines (white) demarcate the zone encompassing the medullary regions. Cross-lines (black) differentiate vessels from hypoxic regions. B: oxygenation level (R2*) in the cortex. Renal artery stenosis (RAS) decreased cortical oxygenation level (increased R2*) in both Lean-STK and ObM-STK groups. C and D: oxygenation level in the medulla and its response to furosemide. Impaired tubular transport function was indicated in ObM-STK. ⽥RAS: significant effect of RAS (2-way ANOVA). *P ⬍ 0.05 vs. Lean-sham; #P ⬍ 0.05 vs. pre-furosemide (paired t-test). AJP-Renal Physiol • doi:10.1152/ajprenal.00043.2013 • www.ajprenal.org

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Table 2. Single-kidney hemodynamics and function in sham and stenotic kidneys (STK) from Lean or ObM pigs (n ⫽ 7 each) Sham

3

Renal volume, mm RBF, ml/min GFR, ml/min

RAS


Lean

ObM

Lean-STK

ObM-STK

Diet

RAS

DietxRAS

74.5 ⫾ 5.6 427.5 ⫾ 27.2 50.5 ⫾ 5.3

90.1 ⫾ 8.0 584.3 ⫾ 95.4 68.8 ⫾ 7.7

43.4 ⫾ 11.9* 238.6 ⫾ 78.3 27.8 ⫾ 9.8*

63.4 ⫾ 9.8† 311.8 ⫾ 62.7† 36.3 ⫾ 7.8†

0.06 0.17 0.11

0.004 0.007 0.003

0.81 0.60 0.54

Values are means ⫾ SE. RBF, renal blood flow; GFR, glomerular filtration rate. *P ⬍ 0.05 vs. Lean-sham. †P ⬍ 0.05 vs. ObM-sham.

was also measured from CT images and expressed as a fraction of the abdominal cavity volume, as described previously (22). BOLD-MRI. Three days before MDCT, 3T BOLD-MRI (Signa Echo Speed; GE Medical Systems, Milwaukee, WI) was performed as described (11, 32) to assess intrarenal oxygenation, expressed as R2*, an index of deoxyhemoglobin concentration in the kidney cortex and medulla. To further examine tubular transport in the medullary thick ascending limb of Henle’s loop, BOLD measurements were repeated 15 min after a furosemide (20 mg) injection into an ear vein cannula. In viable medullary tubules, furosemide inhibits solute transport activity and thereby oxygen demand, and improves medullary oxy-

In Vivo Studies MDCT. At 16 wk, MDCT scanning was performed to assess the STK and contralateral (CLK) kidney volume, RBF and glomerular filtration rate (GFR), as described previously (5, 9). Briefly, 160 consecutive scans were performed following a central venous injection of iopamidol (0.5 ml·kg⫺1·2 s⫺1). Then, MDCT images were reconstructed and displayed with the Analyze software package (Biomedical Imaging Resource, Mayo Clinic, Rochester, MN). For data analysis, regions of interest (ROI) were drawn in the aorta, renal cortex, and medulla, and time-attenuation curves were used to obtain measures of RBF and GFR (9). Intra-abdominal adipose tissue volume

A

Sham

RAS

Lean

Lean-STK

ObM-STK

B 2.00 ♠ RAS

2.00 Inner Cortex

Outer Cortex

Vessels/mm 2

♠ RAS ♠ DietxRAS

1.00

♠ RAS

1.00

*

*

§

§

*

*§

§

0.00

0.00 20-40 µm

C

♠ DietxRAS

40-200 μm

20-40 µm

200-500 µm

40-200 μm 200-500 µm

12

6

0 Lean

ObM

LeanSTK

Sham 1.4

♠ DietxRAS

0.30

*

♠ RAS ♠ DietxRAS

Sham

* 0.7

FLK-1

VEGF

Average vessel diameter (m)

Inner cortex

Outer cortex

ObM

0.0 Lean

ObM

Sham

Lean- ObMSTK STK

RAS

Lean

0.15 §

VEGF 42 kDa

ObM

ObMSTK

RAS

RAS

LeanSTK

ObMSTK

FLK-1 180 kDa GAPDH 37 kDa

0.00 Lean

ObM

Sham

LeanSTK

ObMSTK

RAS

Fig. 2. Microcirculation and angiogenic activities in sham and STK from Lean and ObM pigs (n ⫽ 7 each). A: representative 3-dimensional tomographic images of the cortical microcirculation. B: quantifications of microvascular density in small, medium, and large vessels, and the average vessel diameter. Arrows (green) indicate arcuate arteries at the junction of the renal cortex and medulla. C: renal protein expressions of vascular endothelial growth factor (VEGF) and its receptor-2 FLK-1. Protein bands were quantified relative to GAPDH. Two representative bands from each group are shown. ⽥RAS: significant effect of RAS; ⽥DietxRAS: significant interaction of ObM diet and RAS (2-way ANOVA). *P ⬍ 0.05 vs. Lean-sham; §P ⬍ 0.05 vs. ObM-sham. AJP-Renal Physiol • doi:10.1152/ajprenal.00043.2013 • www.ajprenal.org

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Table 3. Systemic levels of inflammatory markers in sham or RAS pigs with or without ObM (n ⫽ 7 each) Sham

MCP-1, ng/ml TNF-␣, ng/ml sE-selectin, ng/ml IFN-␥, ng/ml IL-17, pg/ml IL-10, pg/ml

RAS


Lean

ObM

Lean

ObM

Diet

RAS

DietxRAS

0.09 ⫾ 0.01 0.03 ⫾ 0.08 6.7 ⫾ 3.7 0.14 ⫾ 0.00 18.81 ⫾ 1.80 8.12 ⫾ 2.81

0.09 ⫾ 0.02 0.06 ⫾ 0.00 4.8 ⫾ 4.8 0.15 ⫾ 0.02 26.82 ⫾ 7.47 6.00 ⫾ 1.34

0.2 ⫾ 0.07* 0.16 ⫾ 0.05* 15.4 ⫾ 7.1 0.55 ⫾ 0.14* 41.49 ⫾ 27.76 4.85 ⫾ 1.60

0.2 ⫾ 0.04*† 0.11 ⫾ 0.03* 36.8 ⫾ 10.8*† 0.40 ⫾ 0.11*† 66.50 ⫾ 36.91 3.71 ⫾ 0.92

0.94 0.60 0.21 0.45 0.48 0.36

0.006 0.009 0.007 0.001 0.18 0.14

0.92 0.16 0.10 0.35 0.71 0.78

Values are means ⫾ SE. MCP-1, monocyte chemotactic protein-1; sE-selectin, soluble E-selectin. *P ⱕ 0.05 vs. Lean-sham. †P ⱕ 0.05 vs. ObM-sham.

Urine microalbumin (mAlb; Arbor Assays, Ann Arbor, MI), creatinine, and a lipid profile (Roche) including total cholesterol, triglycerides, and LDL were assessed. Fasting blood glucose and insulin level were measured by standard procedures, and the homeostasis model assessment of insulin resistance (HOMA-IR) index (fasting plasma glucose ⫻ fasting plasma insulin/22.5) was calculated to evaluate insulin sensitivity (4, 22). Plasma renin activity (RIA; DiaSorin) was measured to evaluate the renin-angiotensin system. Plasma 8-isoprostanes (EIA; Cayman Chemical, Ann Arbor, MI) and oxidized-LDL (Ox-LDL; Alpco Diagnostics, Windham, NH) levels served as systemic oxidative stress indices. Assessments of systemic and kidney release of inflammatory cytokines. Levels of inflammatory cytokines were measured by ELISA in both

genation (decreases R2*) (11). Therefore, the change in R2* is taken as an index of the viability of the tubules. For data analysis, ROIs were manually traced in the cortex and medulla on the 7-ms echo time images that give the best anatomic details in each experimental period. For each echo time, the software automatically computed the average of MR signals within each ROI. The BOLD signal (relaxivity index, R2*) was measured both at baseline and after furosemide, and the change in R2* is presented as delta R2*. Ex Vivo Studies Blood and urine sample. Urine was collected before MDCT by bladder puncture and blood samples from the inferior vena cava (IVC). Sham

A

Sham

C

RAS

Sham 2000

6000 ♠ RAS

5000 0 Lean

ObM

-5000

Lean- ObMSTK STK

4000

-10000

2000 0 Lean

ObM

-2000

LeanSTK

ObMSTK

RAS

♠ Diet

1500

sE-selectin (µg/min)

*

10000

MCP-1 (ng/min)

B

TNF-alpha (ng/min)

15000

RAS

*

*

1000 500 0 Lean

ObM

-500

LeanSTK

ObMSTK

-1000

-4000 -1500

-15000 Sham ♠ RAS

*

10 0 Lean -10

Sham

RAS

F

ObM

LeanSTK

ObMSTK

Sham

RAS

500

1.5

20

IF-γ (ng/min)

E

RAS

-2000

1.0 0.5 0.0 Lean -0.5

ObM

LeanSTK

ObMSTK

IL-10 (pg/min)

30

IL-17 (pg/min)

D

-6000

250

0 Lean

ObM

LeanSTK

ObMSTK

-250 -20

-1.0

-30

-1.5

-500

Fig. 3. Kidney release of inflammatory cytokines in sham and STK from Lean and ObM pigs (n ⫽ 7 each group). A: MCP-1. B: TNF-␣. C: sE-selectin. D: IFN-␥. E: IL-17. F: IL-10. ObM-STK group showed magnified inflammation in the kidney. ⽥RAS: significant effect of RAS; ⽥Diet: significant effect of ObM diet (2-way ANOVA). *P ⱕ 0.05 vs. Lean-sham. AJP-Renal Physiol • doi:10.1152/ajprenal.00043.2013 • www.ajprenal.org

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the IVC and STK renal veins (RV), including MCP-1 (VS0081S-002, Kingfisher Biotech), TNF-␣ (KSC3011 Invitrogen), IFN-␥ (VS0259S-002, Kingfisher), soluble E-selectin (sE-selectin, P4988, Biotang), IL-17 (VS0260S-002, Kingfisher), and the anti-inflammatory marker IL-10 (KSC0101, Invitrogen). The gradient of these cytokines between RV and IVC was assessed, and their net release from the STK was calculated [(RV-IVC) X RBF] (12). Animals were euthanized after a 3-day recovery with a lethal intravenous dose of 100 mg/kg pentobarbital sodium (Sleepaway, Fort Dodge Laboratories, Fort Dodge, IA). Both kidneys were removed using a retroperitoneal incision and immediately dissected and preserved for micro-CT (renal microvasculature) and tissue studies (Western blotting and histology). Abdominal and perirenal fat were excised and prepared for ex vivo studies. Micro CT. After the kidney was flushed, microfil MV122 (an intravascular contrast agent) was perfused into the STK under physiological pressure through a cannula ligated in the renal artery. Samples were prepared and scanned at 0.5° angular increments at 18-␮m resolution, and images were analyzed as previously described (39). The spatial density of microvessels (defined as diameters ⬍500 ␮m) in the inner and outer halves of the renal cortex were calculated using Analyze and classified

according to diameter as small (20 – 40 ␮m), medium (40 –200 ␮m), or large (200 –500 ␮m) microvessels (39). Histology and Western blotting. Intrarenal inflammation was assessed by macrophage (MØ) staining (CD163, 1:50, Abcam). Their subpopulations were evaluated by coexpression of either inducible nitric oxide synthase (iNOS) for proinflammatory M1-MØ or arginase-1 (1:50, both Abcam) for M2-MØ, which resolve inflammation and elicit tissue repair (19, 29). Intrarenal oxidative stress was assessed by Ox-LDL staining (1:50, Abcam) and Western blotting for p47 (1:200, Santa Cruz Biotechnology), and angiogenic activity by expression of vascular endothelial growth factor (VEGF) and its receptor-2 FLK-1 (1:200, both Santa Cruz Biotechnology). Kidney fibrosis was examined by trichrome staining and glomerulosclerosis score (% of sclerotic of 100 glomeruli) (5, 6), and oil-red-O staining for kidney fat deposition. Abdominal and perirenal fat tissues were also examined for inflammation by M1-MØ and TNF-␣ (1:50, Abcam) staining (30) and fibrosis by trichrome. Histochemical analysis utilized a computer-aided image-analysis program (AxioVision, Carl Zeiss MicroImaging, Thornwood, NY). GAPDH (1:5,000 Covance, Emeryville, CA) served as a loading control for Western blotting.

Sham

A

RAS

Lean

Lean-STK

ObM

ObM-STK

M1-MØ (iNOS/CD163)

10 μm

M2-MØ (Arginase/CD163)

10 μm

3

5

C

♠ Diet

*

†

♠ Diet ♠ RAS

§

*

2

M1/M2 ratio

M1-MØ (positive cell %)

B

* 1

3

0

0 Lean Sham

ObM

LeanSTK RAS

ObMSTK

Lean

ObM Sham

LeanSTK

ObMSTK RAS

Fig. 4. Macrophage (MØ) phenotype in the kidney of sham and RAS with or without ObM (n ⫽ 7 each group). A: representative images (⫻40) of immunofluorescence staining for M1- and M2-MØ [CD163 red, M1 (inducible nitric oxide synthase; iNOS) or M2 (arginase-1) green, double staining yellow]. B: quantification of M1-MØ-positive cells. C: M1/M2 ratio phenotype subpopulations. ObM-STK exhibited amplified proinflammatory MØ infiltration. ⽥RAS: significant effect of RAS; ⽥Diet: significant effect of the ObM diet (2-way ANOVA). *P ⬍ 0.05 vs. Lean-sham; §P ⬍ 0.05 vs. ObM-sham; †P ⬍ 0.05 vs. Lean-STK. AJP-Renal Physiol • doi:10.1152/ajprenal.00043.2013 • www.ajprenal.org

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Statistical Analysis Statistical analysis was performed using JMP software package version 9.0 (SAS Institute, Cary, NC). Results are expressed as means ⫾ SE. Two-way ANOVA was used to analyze the effects of RAS and diet as separate factors, and their interactions, followed by Tukey’s test as appropriate. A paired Student’s t-test was performed for comparisons within groups (BOLD medullary R2* before and after furosemide; function of STK vs. CLK). Results were considered significant for P ⱕ 0.05. RESULTS

Animal Characteristics At 16 wk, ObM groups had higher body weight and visceral obesity, reflected by an increased abdominal fat volume fraction (Table 1). RAS groups had increased mean arterial pressure (MAP) in both Lean and ObM compared with their relative controls. The ObM diet had a stimulating effect on MAP but did not significantly increase MAP in either RAS or sham (both P ⬎ 0.10). ObM but not

A

†

Ox-LDL levels (μ/L)

ObM

Lean-STK

ObM-STK

Sham Lean

ObM

LeanRAS

ObMRAS

RAS

*

♠ Diet

Ox-LDL Staining

0

†

* 20

Sham

D

Lean

0 Lean

ObM

Sham 4

♠ RAS

LeanRAS

RAS

*

ObMRAS

RAS

ObM

ObMSTK

LeanSTK

P47 47 kDa GAPDH 37 kDa 0.8

*

§ †

♠ Diet ♠ RAS ♠ DietxRAS

*

2

P47

Ox-LDL expression (area%)

Renal

C

100 μm

Lean

*

♠ Diet ♠ RAS

Sham

Systemic

RAS reduced volume and GFR in Lean-STK, suggesting decreased blood supply and renal function (Table 2). RAS also decreased RBF and GFR in ObM-STK compared with its sham control but not compared with Lean-sham (all P ⬎ 0.20), suggesting the blood supply and function in ObM-STK were

B

125

40

Renal Hemodynamics, Function and Oxygenation

RAS

Systemic

8-Isoprostane levels (pg/ml)

250

Lean groups also developed hyperinsulinemia and insulin resistance (HOMA-IR), as well as elevated triglyceride and LDL levels. Collectively, this cluster of abnormalities suggested the development of obesity-metabolic syndrome in ObM groups. In addition, RAS increased urine concentration of mAlb in both Lean and ObM (P ⬎ 0.50 ObM-RAS vs. Lean-RAS) and had an effect on increasing plasma creatinine regardless of diet, although it was not elevated in either group. The ObM diet had no effect on mAlb or plasma creatinine. The ObM diet and RAS elevated PRA only in the ObM-RAS group (P ⫽ 0.01 vs. Lean-sham). No interaction between ObM and RAS was found for these parameters.

0.4

0 Lean

ObM

LeanSTK

ObMSTK

0.0 Lean

Sham

ObM

RAS

LeanSTK

ObMSTK

Fig. 5. Systemic and local oxidative stress in sham and RAS pigs with or without ObM (n ⫽ 7 each group). A: systemic 8-isoprostanes and oxidized-low density lipoprotein (Ox-LDL) levels. B and C: representative images (⫻20) and quantification of kidney Ox-LDL expression. D: renal protein expression of p47 quantified relative to GAPDH. Two representative bands from each group are shown. ObM-RAS exhibited enhanced oxidative stress in both circulation and the STK. ⽥Diet: significant effect of the ObM diet; ⽥RAS: significant effect of RAS; ⽥DietxRAS: significant interaction of the ObM diet and RAS (2-way ANOVA). *P ⬍ 0.05 vs. Lean-sham; §P ⬍ 0.05 vs. ObM-sham; †P ⬍ 0.05 vs. Lean-RAS. AJP-Renal Physiol • doi:10.1152/ajprenal.00043.2013 • www.ajprenal.org

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relatively preserved compared with normal pigs. RAS elevated BOLD-MRI cortical R2* in both Lean-STK and ObM-STK compared with Lean-sham, indicating lower cortical oxygenation (Fig. 1). In the medulla, although the basal R2* was unaffected by either RAS or diet, only ObM-STK revealed a diminished R2* response to furosemide (P ⫽ 0.10 for Post-furosemide vs. Prefurosemide, Fig. 1C). In addition, RAS decreased delta R2* only in ObM-STK (P ⫽ 0.03 vs. Lean-sham) but not Lean-STK (Fig. 1D). These data indicated impaired medullary tubular oxygendependent transport activity in ObM-STK.

Consistent with these findings, the ObM diet and RAS also interacted to downregulate the expression of the angiogenic factor VEGF and further decreased its receptor FLK-1 in ObM-STK compared with sham control (P ⫽ 0.002). In contrast, Lean-STK increased VEGF, and ObM-sham elevated FLK-1 (Fig. 2C). Hence, the coexistence and interaction of the ObM diet and RAS mediate intrarenal microvascular remodeling in ObM-STK by suppressing neovascularization and blunting angiogenic activity.

Intrarenal Microcirculation

The levels of systemic inflammatory markers are presented in Table 3. RAS elevated in both Lean and ObM the levels of MCP-1, TNF-␣, and IFN-␥. In addition, RAS also elevated sE-selectin in ObM but not Lean. The ObM diet had no effect on systemic levels of these cytokines. IL-17 and anti-inflammatory IL-10 levels were unchanged. In the kidney, RAS elevated MCP-1 and IFN-␥ only in ObMSTK but had no effect on Lean-STK. The ObM diet elevated sE-selectin in both sham and STK (Fig. 3). The release of IL-17 and IL-10 were again unchanged. Hence, RAS mainly activated

While there was no significant effect of RAS on the vessel density in Lean-STK, there was a decrease in ObM-STK in the density of both small (20 – 40 ␮m) and large vessels (200 –500 ␮m) in both the outer and inner cortex (vs. sham control). Of note, the ObM diet interacted with RAS to aggravate its effect on small vessels (Fig. 2, A and B). In contrast, ObMsham enhanced neovascularization in these vessels. The average vessel diameter was unchanged.

A

Inflammation

Sham

RAS

Lean

Lean-STK

ObM-STK

100 μm

Oil-Red-O

ObM

100 μm

Trichrome

B

†§

♠ Diet ♠ RAS

0.5

0.0 Lean

12

*

1.0

Oil red O (area %)

D

ObM

Sham

LeanSTK

ObMSTK

RAS

E ♠ RAS

*

6

*

0 Lean

ObM

Sham

LeanSTK

ObMSTK

Global glomerulosclerosis (%)

1.5

(Trichrome area%)

C

30

†

♠ Diet ♠ RAS ♠ DietxRAS

*

20

10

0

RAS

Lean

ObM

Sham

LeanSTK

ObMSTK

RAS

Fig. 6. Renal fat deposition and fibrosis in sham and STK from Lean and ObM pigs (n ⫽ 7 each group). A and C: representative kidney oil-red-O staining images (⫻20) and its quantification. B, D, and E: representative trichrome images (⫻20) and quantifications of tubulointerstitial fibrosis and sclerotic glomeruli. ⽥Diet: significant effect of ObM diet; ⽥RAS: significant effect of RAS; ⽥DietxRAS: significant interaction of ObM diet and RAS (2-way ANOVA). *P ⬍ 0.05 vs. Lean-sham; §P ⬍ 0.05 vs. ObM-sham; †P ⬍ 0.05 vs. Lean-STK. AJP-Renal Physiol • doi:10.1152/ajprenal.00043.2013 • www.ajprenal.org

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inflammatory cytokines in the systemic circulation, but ObM-RAS magnified inflammation in both the systemic circulation and STK. Furthermore, the ObM diet amplified recruitment of M1-MØ in both RAS and sham compared with Lean-sham. Importantly, the ObM diet and RAS increased (albeit not synergistically) the M1/M2-MØ ratio in ObM-STK (P ⫽ 0.0004 vs. Lean-sham, P ⫽ 0.018 vs. ObM-sham, and P ⫽ 0.0034 vs. Lean-STK, Fig. 4), an index of the proinflammatory phenotype shift of MØ, while there was no effect of RAS on the M1/M2-MØ ratio in Lean-STK. Therefore, in addition to elevating inflammatory cytokines in the kidney, ObM-STK also enhanced recruitment of proinflammatory M1-MØ, which is associated with the effect of the ObM diet.

0.001 vs. ObM-sham, and P ⫽ 0.004 vs. Lean-STK, Fig. 5D), whereas Lean-STK was not affected. Hence, the coexistence of the ObM diet and RAS also induced prominent oxidative stress in the ObM-RAS group in both systemic circulation and the STK. The ObM diet and RAS induced distinct fat deposition only in ObM-STK, revealed by oil-red-O staining (P ⫽ 0.002 vs. Lean-sham, P ⫽ 0.03 vs. ObM-sham, and P ⫽ 0.0007 vs. Lean-STK, Fig. 6, A and C). The RAS increased tubulointerstitial fibrosis in both STKs (Fig. 6, B and D), but the ObM diet and RAS further synergistically increased the number of sclerotic glomeruli only in ObM-STK (P ⫽ 0.01 vs. Lean-sham and P ⫽ 0.02 vs. Lean-STK, Fig. 6).

Oxidative Stress, Fat Deposition, and Renal Fibrosis

Fat Inflammation and Fibrosis

The ObM diet and RAS increased the level of 8-isoprostane in ObM-RAS, and both ObM groups had elevated Ox-LDL (Fig. 5A), whereas Lean-RAS was not affected. In the kidney, the RAS increased the expression of Ox-LDL in both STKs (both P ⬍ 0.01 vs. Lean, Fig. 5, B and C). However, ObM-STK also showed increased expression of p47, attributed to the effects of the ObM diet, RAS, and their interaction (P ⫽ 0.002 vs. Lean-sham, P ⫽

Both ObM groups showed increased infiltration of M1-MØ, expression of TNF-␣, and fibrosis in abdominal fat compared with Lean-sham (Fig. 7, A–D). Similarly, in the perirenal fat, ObM also elevated infiltration of M1-MØ, and in the ObMSTK, TNF-␣ expression as well (Fig. 7, E and F). The RAS also showed a stimulating effect on M1-MØ infiltration, although their number was not elevated in Lean-STK. These data

50 μm

TNF-alpha

B M1-MØ (positive cell %)

50 μm

♠ Diet 30

*

*

15

0

TNF-alpha (area %)

M1-MØ

Abdominal Fat

A

3

1.5

0 Lean ObM Lean- ObMRAS RAS Sham RAS

Lean ObM Lean- ObMRAS RAS Sham RAS

C

Abdominal Fat Trichrome ObM Lean

Lean-RAS

ObM-RAS

100 μm

*

*

D

5

(area %)

♠ Diet

* † 2.5

*

0 Lean

ObM

M1-MØ

TNF-alpha

F M1-MØ (positive cell %)

Perirenal Fat

E

40

*†

♠ Diet ♠ RAS

*

20

0 Lean

ObM Lean- ObMSTK STK

Sham

RAS

TNF-alpha (area %)

Sham 6

♠ Diet

Lean RAS

ObM RAS

RAS

*†

3

0 Lean ObM Lean- ObMSTK STK Sham RAS

Fig. 7. MØ, inflammation, and fibrosis in the abdominal and perirenal adipose tissues of sham and RAS pigs with or without ObM (n ⫽ 7 each group). A and B: representative images (⫻40) of immunofluorescence staining for M1-MØ (CD163 red, M1-iNOS green, double staining yellow) and TNF-␣ (green) and their quantifications. C and D: representative images (⫻20) of trichrome staining in abdominal fat, and its quantification. E and F: representative images (⫻40) of M1-MØ and TNF-␣ in perirenal fat. ObM diet enhanced M1-MØ and TNF-␣ expression in both abdominal and perirenal fat. ⽥Diet: significant effect of ObM diet; ⽥RAS: significant effect of RAS (2-way ANOVA). *P ⱕ 0.05 vs. Lean-sham; †P ⱕ 0.05 vs. Lean-STK. AJP-Renal Physiol • doi:10.1152/ajprenal.00043.2013 • www.ajprenal.org

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Table 4. Single-kidney hemodynamics and function in sham and contralateral kidneys (CLK) of RAS pigs with or without ObM (n ⫽ 7 each) Sham

Renal volume, mm3 RBF, ml/min GFR, ml/min

RAS


Lean

ObM

Lean-CLK

ObM-CLK

Diet

RAS

DietxRAS

74.5 ⫾ 5.6 427.5 ⫾ 27.2 50.5 ⫾ 5.3

90.1 ⫾ 8.0 584.3 ⫾ 95.4 68.8 ⫾ 7.7

89.8 ⫾ 6.3 530.9 ⫾ 40.8 62.5 ⫾ 3.6

121.5 ⫾ 5.4*† 626.9 ⫾ 42.9* 80.3 ⫾ 7.5*†

0.001 0.045 0.005

0.001 0.23 0.14

0.22 0.61 0.72

Values are expressed as means ⫾ SE. *P ⬍ 0.05 vs. Lean-sham. †P ⬍ 0.05 vs. Lean-CLK.

CLKs Both ObM-CLK and Lean-CLK showed increased volume, RBF, and GFR compared with their corresponding STKs (all P ⬍ 0.01). Furthermore, the ObM diet magnified these parameters in

A

Lean-CLK

ObM-CLK

100 μm

B

the CLK compared with Lean-sham (all P ⬍ 0.01) and its GFR compared with Lean-CLK, indicating hyperfiltration in ObMCLK (Table 4). The RAS increased renal oxidative stress in the CLKs, reflected by elevated Ox-LDL in Lean-CLK and the expression of p47 in ObM-CLK (data not shown). Additionally, similar to the STK, the ObM diet also enhanced CLK fat deposition (both P ⬍ 0.01 vs. Lean-sham and vs. Lean-CLK, Fig. 8A), infiltra-

ObM-CLK

Lean-CLK

10 μm

M1-MØ

♠ Diet

1

0 ObM

Sham

LeanCLK

♠ Diet

2

* 0 Lean

ObM Lean- ObMCLK CLK

2

*

Lean ObM Lean- ObMCLK CLK

RAS 4

100 µm

♠ Diet ♠ RAS

§

* *

2

0 Lean

RAS

1.0

ObM

Sham

Lean- ObMCLK CLK RAS

Global glomerulosclerosis (%)

ObM-CLK

*

0

ObMCLK

Trichrome (area%)

Trichrome

Lean-CLK

RAS

♠ Diet

Sham

C

*

1

4

M2-MØ

*

Lean

3

Sham

†

M1/M2 ratio

Oil red O (area %)

2

M1 MØ (positive cell %)

suggest the ObM diet had enhanced inflammatory activity not only in abdominal adipose tissue but also in peripheral fat, particularly around the STK.

0.5

0.0 Lean ObM Lean- ObMCLK CLK Sham RAS

Fig. 8. Renal fat deposition, MØ phenotype, and fibrosis in sham and the contralateral kidneys (CLK) of Lean and ObM pigs with renal artery stenosis (n ⫽ 7 each group). A: representative kidney oil-red-O staining images (⫻20) and its quantification showed increased fat deposition in ObM-CLK. B: representative images (⫻40) of immunofluorescence staining for M1- and M2-MØ [CD163 red, M1-(iNOS) or M2 (arginase-1) green, double staining yellow] and quantifications for M1-MØ-positive cells and the M1/M2-MØ ratio. As opposed to Fig. 4, the increase in the M1/M2 ratio in ObM-sham reached statistical significance because of the different groups involved in analysis. C: representative trichrome images (⫻20) and quantifications for tubulointerstitial fibrosis and glomerulosclerosis. ⽥Diet: significant effect of the ObM diet; ⽥RAS: significant effect of RAS (2-way ANOVA). * P ⱕ 0.05 vs. Lean-sham; §P ⬍ 0.05 vs. ObM-sham; †P ⬍ 0.05 vs. Lean-CLK. AJP-Renal Physiol • doi:10.1152/ajprenal.00043.2013 • www.ajprenal.org

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tion of M1-MØ, and the M1/M2 ratio (Fig. 8B). Both CLKs had mild but detectable tubulointerstitial fibrosis, but neither developed significant glomerulosclerosis (Fig. 8C). Therefore, the RAS induced mild oxidative stress and fibrosis in the CLK, while the ObM diet induced hyperfiltration and increased fat deposition and M1-MØ infiltration. Therefore, the coexistence of ObM resulted in greater injury in RAS-CLK. DISCUSSION

This study shows that obesity associated with metabolic derangements, in addition to amplifying inflammation, interacts with renal artery stenosis to increase oxidative stress, impairs tubular function, blunts microvascular angiogenesis, and accelerates glomerulosclerosis in the STK, although at this early stage its GFR and RBF were relatively preserved. In the contralateral kidney, ObM also induces hyperfiltration and mediates fibrosis. Thus the constellation of the metabolic syndrome constitutes a risk factor for bilateral kidney structural damage in unilateral renovascular disease. The proinflammatory effects of obesity on the kidney have been proposed previously (17, 26), yet its impact on the kidney subjected to chronic ischemia remained unidentified. In the present study, the coexistence of ObM and RAS magnified not only endothelial activation in the systemic circulation compared with RAS alone (sE-selectin), but also kidney release of inflammatory cytokines (MCP-1, sE-selectin, and IFN-␥), and in the bilateral kidneys induced infiltration of macrophages and MØ phenotype shift, which Lean-RAS did not. This observation extends findings in other forms of CKD on the contribution of a proinflammatory microenvironment to kidney injury (31), and underscores a key association of MØ infiltration and inflammatory phenotype with renal pathology in the ObMcomplicated RAS. However, it is important to note that although the activated cytokines in ObM-STK were mainly attributed to the effect of the RAS, the RAS did not increase

their release from Lean-STK. In addition, M1-MØ infiltration in the kidney was attributed only to the ObM diet. It is thus likely that during coexistence of ObM-RAS, the ObM diet plays a major role in kidney inflammation, both by directly amplifying M1-MØ and by facilitating the effect of RAS. Furthermore, adipose tissue contains adipocytes which are recognized as cytokine-secretory cells (36), including MØ that prevail in obesity (23, 35), and abundant inflammatory cytokines like TNF-␣ (34), as observed in the present study (abdominal and perirenal fat). Interestingly, our study also found that the ObM diet elevated M1-MØ in the STK, in parallel to its effect in the fat tissue, whereas the RAS had not effect on M1-MØ. Speculatively, increased M1-MØ and TNF-␣ activity in adjacent perirenal fat may potentially contribute to inflammation and MØ recruitment in the STK. On the other hand, elevated PRA in ObM-RAS may also reflect the contribution of angiotensin II to MØ activation and amplified inflammation via binding to its receptor on MØ (24). Further studies will be needed to elucidate the cross talk between the STK and its perirenal fat. Our previous study has shown that early obesity increased intrarenal microvascular proliferation and angiogenic activity (22). In the current study, we observed the similar effect in the ObM-sham group. The Lean-STK exhibited relatively preserved microvascular density, possibly due to a compensatory increase in VEGF in response to ischemia (13). However, the ObM diet interacted with the RAS and blunted in ObM-STK both neovascularization and angiogenic signaling. This finding suggests that microvessels spawned in ObM may be fragile and prone to loss (20). Additionally, prolonged and severe inflammation and oxidative stress interfere with upregulation of angiogenic factors (7, 8, 39). Indeed, suppressed microcirculation in ObM-STK was accompanied by marked inflammation (MCP-1, IFN-␥, and MØ) and magnified oxidative stress, which may explain the suppressed

Obesity-Metabolic Derangement

Other factors

Insulin resistance

Adipocytes

Renal Artery Stenosis

Lipid overload Renin-Angiotensin system

Hemodynamic alteration

Fig. 9. Potential mechanisms mediating kidney injuries in renovascular disease with obesity-metabolic derangements. ROS, reactive oxygen species; Ox-LDL, oxidized-low density lipoprotein; FFA, free fatty acid; HBP, hypertension.

a

Intracellular lipid

Hyperfiltration

Albuminuria (FFA enriched)

Glomerular injury

ROS or Ox-LDL

TNF-α, IF- , MCP-1, M1-MØ

Tubular injury

Oxidative stress

Endothelial activation (sE-selectin)

Tubulo-interstitial fibrosis Glomerulosclerosis

AJP-Renal Physiol • doi:10.1152/ajprenal.00043.2013 • www.ajprenal.org

Vasoconstriction

Microvasculature remodeling


vessel density in ObM-STK that was not observed in ObM or the RAS alone. Therefore, the coexistence of ObM and the RAS may predispose the kidney to microvascular regression through amplified inflammation and oxidative stress. Given the suppressed neovascularization ex vivo with no change in diameter, the relatively preserved RBF in the ObMSTK is unlikely due to microvascular remodeling. Rather, hemodynamic factors like increased cardiac output and blood volume induced by ObM may possibly lead to vasodilation and a rise in RBF in vivo. In contrast, Lean-STK had decreased RBF but relatively preserved microcirculation. Possibly as a result, ObM-STK and Lean-STK developed similar degrees of hypoxia (R2* by BOLD MRI), yet tubular injury (diminished response to furosemide) and renal scarring (glomerulosclerosis) were more pronounced in the ObM-STK. Reflected by oil-red-O staining, the ObM diet increased fat deposition in ObM-STK. Because free fatty acids bind to albumin (31), enhanced albumin excretion by the RAS may facilitate their excess glomerular filtration that is not apparent in Lean-STK and enhance their reabsorption by the renal proximal tubules, resulting in tubulointerstitial inflammation and fibrosis (18, 31). Through similar pathways (31, 33), increased renal fat deposition may also lead to glomerular injury, such as mesangial cell proliferation and extracellular matrix deposition (1). In line with this notion, the present study found that ObM synergized with the RAS to increase glomerulosclerosis. The current study shows that obesity-metabolic derangement augments renal hemodynamics, reflected by preserved RBF and GFR in the STK and hyperfiltration in the CLK. Nevertheless, increased glomerulosclerosis and suppressed neovascularization in the ObM-STK may expose the remaining nephrons to greater risk of hyperfiltration, thereby accelerating their loss, following a similar pattern observed in the progression of chronic renal failure (25). This notion is supported by the observation of greater glomerulosclerosis in the ObM-STK than Lean-STK. In addition, increased glomerular filtration pressure may also facilitate fat deposition in the CLK and subsequent adverse effects. Therefore, the bolstered hemodynamics in ObM may not protect the kidney from the insult of renovascular disease but rather accelerate its structural damage. Indeed, STK of patients with ObM are less likely to improve after revascularization (10), possibly due to greater irreversible damage compared with non-ObM patients. Limitation Our study is limited by the use of relatively young animals and the short duration of the disease, yet renal structure and function in our swine model are similar to humans. Furthermore, this model recapitulates many features of the metabolic syndrome in humans. Further studies will need to pursue individual pathways and longer durations of kidney injury to determine its reversibility in ObM-RAS. In addition, the possible effects of ObM on the progression of degree of RAS, the effectiveness of treatments like revascularization, and identification of specific factors influencing renal outcomes warrant further exploration. Conclusion As summarized in Fig. 9, our study suggests that obesity associated with metabolic derangements exerts complex roles in mediating kidney damage distal to renovascular disease,

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including aggravated inflammation that involves M1-MØ and related cytokines, blunted microcirculation, enhanced oxidative stress, and accelerated scarring in the kidney. These pathological alterations might be related to magnified fat deposition in the kidney and activated MØ in the perirenal fat tissue attributable to the ObM diet. Additionally, ObM not only directly induces injury in the kidney but also synergizes and facilitates the adverse effects of RAS on microvascular remodeling, glomerular damage, and tubular dysfunction. The preserved function in the ObM-STK may also permit disease progression in remaining nephrons. In conclusion, MØ-accentuated inflammatory renal pathways may constitute important mechanisms and potential therapeutic targets in addition to restoring blood flow in ObM-RAS kidneys. Prevention of obesity and its consequence likely will prove to be important strategies to combat the kidney injury subject to chronic ischemia. GRANTS This study was partly supported by National Institutes of Health Grants DK73608, HL77131, HL085307, C06-RR018898, and UL1 TR000135. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: X.Z., S.C.T., and L.O.L. provided conception and design of research; X.Z., J.R.W., A.E., B.E., J.A.C., X.Y.Z., A.P., J.D.K., K.L.J., and H.T. performed experiments; X.Z., Z.-L.L., J.R.W., A.E., B.E., J.A.C., X.Y.Z., A.P., J.D.K., K.L.J., and H.T. analyzed data; X.Z., Z.-L.L., A.E., B.E., J.A.C., X.Y.Z., A.P., J.D.K., K.L.J., H.T., S.C.T., A.L., and L.O.L. interpreted results of experiments; X.Z. and J.A.C. prepared figures; X.Z. drafted manuscript; X.Z., S.C.T., A.L., and L.O.L. edited and revised manuscript; X.Z., Z.-L.L., J.R.W., B.E., J.A.C., X.Y.Z., A.P., J.D.K., K.L.J., H.T., S.C.T., A.L., and L.O.L. approved final version of manuscript. REFERENCES 1. Abrass CK. Cellular lipid metabolism and the role of lipids in progressive renal disease. Am J Nephrol 24: 46 –53, 2004. 2. Bagby SP. Obesity-initiated metabolic syndrome and the kidney: a recipe for chronic kidney disease? J Am Soc Nephrol 15: 2775–2791, 2004. 3. Bonnet F, Deprele C, Sassolas A, Moulin P, Alamartine E, Berthezene F, Berthoux F. Excessive body weight as a new independent risk factor for clinical and pathological progression in primary IgA nephritis. Am J Kidney Dis 37: 720 –727, 2001. 4. Chade AR, Krier JD, Galili O, Lerman A, Lerman LO. Role of renal cortical neovascularization in experimental hypercholesterolemia. Hypertension 50: 729 –736, 2007. 5. Chade AR, Rodriguez-Porcel M, Grande JP, Krier JD, Lerman A, Romero JC, Napoli C, Lerman LO. Distinct renal injury in early atherosclerosis and renovascular disease. Circulation 106: 1165–1171, 2002. 6. Chade AR, Rodriguez-Porcel M, Herrmann J, Zhu X, Grande JP, Napoli C, Lerman A, Lerman LO. Antioxidant intervention blunts renal injury in experimental renovascular disease. J Am Soc Nephrol 15: 958 –966, 2004. 7. Chade AR, Zhu X, Lavi R, Krier JD, Pislaru S, Simari RD, Napoli C, Lerman A, Lerman LO. Endothelial progenitor cells restore renal function in chronic experimental renovascular disease. Circulation 119: 547– 557, 2009. 8. Chade AR, Zhu X, Mushin OP, Napoli C, Lerman A, Lerman LO. Simvastatin promotes angiogenesis and prevents microvascular remodeling in chronic renal ischemia. FASEB J 20: 1706 –1708, 2006. 9. Daghini E, Primak AN, Chade AR, Krier JD, Zhu XY, Ritman EL, McCollough CH, Lerman LO. Assessment of renal hemodynamics and function in pigs with 64-section multidetector CT: comparison with electron-beam CT. Radiology 243: 405–412, 2007. 10. Davies MG, Saad WE, Bismuth J, Naoum JJ, Peden EK, Lumsden AB. Impact of metabolic syndrome on the outcomes of percutaneous renal angioplasty and stenting. J Vasc Surg 51: 926 –932, 2010.


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