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Am J Physiol Renal Physiol 293: F1325-F1331, 2007. First published August 8, 2007; doi:10.1152/ajprenal.00039.2007
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Effect of uremia on HDL composition, vascular inflammation, and atherosclerosis in wild-type mice

Christian A. Bang,1 Susanne Bro,1,2 Emil D. Bartels,1 Tanja X. Pedersen,1 and Lars B. Nielsen1,3

Departments of 1Clinical Biochemistry and 2Nephrology, Rigshospitalet, and 3Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

Submitted 22 January 2007 ; accepted in final form 24 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Wild-type mice normally do not develop atherosclerosis, unless fed cholic acid. Uremia is proinflammatory and increases atherosclerosis 6- to 10-fold in apolipoprotein E-deficient mice. This study examined the effect of uremia on lipoproteins, vascular inflammation, and atherosclerosis in wild-type C57BL/6J mice. Uremia was induced by nephrectomy (NX) and increased plasma urea and creatinine concentrations 2.5- to 4.5-fold; control mice were sham operated. After NX, mice were fed a Western-type diet or the same diet with 0.5% cholic acid. Cholic acid-fed NX mice did not thrive and were killed. In NX mice fed the Western-type diet (n = 7), the total plasma cholesterol concentration was similar to that in sham mice (n = 11), but on gel filtration the LDL/HDL cholesterol ratio was increased. HDL from NX mice contained more serum amyloid A and triglycerides and less cholesterol than HDL from sham mice. Plasma concentrations of sICAM-1 and sVCAM-1 and aortic mRNA expression of ICAM-1 and VCAM-1 did not differ between NX and sham mice. Twenty-six weeks after NX, the average oil red O-stained area of the aortic root was similar in NX and sham mice fed the Western-type diet, while it was increased in cholic acid-fed sham mice. The results suggest that moderate uremia neither induces aortic inflammation nor atherosclerosis in C57BL/6J mice despite increased LDL/HDL cholesterol ratio and altered HDL composition.

inflammation; serum amyloid A

CHRONIC RENAL FAILURE INCREASES the risk of cardiovascular disease (11, 12). Although often encountered in uremic patients, hypercholesterolemia, hypertension, and smoking cannot explain the increased risk (9). However, less firmly established risk factors such as increased plasma concentrations of triglyceride-rich apolipoprotein B-containing lipoproteins (1, 18) (with normal total plasma cholesterol concentrations) and inflammation (18) may play a role. In addition, the maturation of HDL seemingly is a defect implying that uremia could impair the otherwise cardioprotective effect of HDL (31).

Moderate uremia (induced by nephrectomy) leads to markedly increased atherosclerosis in hypercholesterolemic apolipoprotein E (apoE)-deficient mice (3–7, 15, 20). This effect cannot be ascribed to changes in the total plasma cholesterol level (3). Nevertheless, in apoE-deficient mice uremia confers increases in the expression of inflammatory genes in the arterial wall (5, 6), suggesting that inflammation could play a role. Interestingly, systemic inflammation has been suggested to convert HDL from a protective to a harmful lipoprotein with proatherogenic effects (30). This effect has been suggested to be related to the increase in serum amyloid A (SAA) plasma concentrations that occurs during inflammation (22, 30). SAA is an amphipathic protein that can incorporate in HDL (10, 14). SAA-enriched HDL is believed to have proatherogenic effects, and SAA has been detected in atherosclerotic lesions where it colocalizes with apoA-I (25). It is unknown, however, whether SAA-enriched HDL may be formed during uremia and how it may affect development of atherosclerosis. The apoE-deficient mouse model may not be suited to address this issue, since their plasma lipoprotein composition is dominated by a vast accumulation of atherogenic apoB48-containing lipoproteins in plasma (34).

Mice normally have very low plasma levels of apoB-containing VLDL and LDL, with the major portion of the plasma cholesterol being in HDL. Nevertheless, the inbred C57BL/6J mouse strain is susceptible to development of fatty-streak-like atherosclerotic lesions when fed a plasma cholesterol-raising cholic acid-containing diet (24, 27), whereas lesion formation occurs but is minimal when the mice are fed a Western-type diet enriched with cholesterol and fat (29). Interestingly, dietary cholic acid raises plasma SAA concentrations (26), although the importance of this effect in relation to atherosclerosis is unclear (17, 28). Two previous studies have examined atherosclerosis in nephrectomized C57BL/6J mice with moderate uremia. One study showed that uremic mice indeed can develop oil-red-O-stained lesions when fed a cholic acid-free, cholesterol- and fat-enriched diet (29). A more recent study noted that three of eight uremic wild-type mice had aortic lesions 12 wk after nephrectomy when fed a low-cholesterol, low-fat diet (7).

In this study, we sought to examine to what extent moderate uremia might affect plasma lipoproteins, vascular inflammation, and development of atherosclerosis in C57BL/6J mice. For this purpose, we examined plasma lipids and lipoprotein profiles, SAA, hepatic expression of genes involved in synthesis of apoB- and apoA-I containing lipoproteins, and aortic expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) mRNA, as well as nitrotyrosine staining and lesion formation in aortas of uremic and control mice fed a cholesterol- and fat-enriched diet and in mice fed a cholic acid-containing diet.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. C57BL/6J wild-type mice (Taconic M&B Laboratory Animals and Services for Research, Ry, Denmark) were housed in a temperature-controlled facility (21–23°C) with a 12:12-h light-dark cycle. The mice were allowed free access to food and water. The experiments were performed according to the principles stated in the Danish law on animal experiments and were approved by the Animal Experiments Inspectorate, Ministry of Justice, Denmark. Before induction of uremia, the mice were fed a standard mouse chow (Altromin 1314, Altromin, Lage, Germany). After induction of uremia or sham operation, mice were switched to a high-cholesterol/high-fat diet (C13002 [GenBank] , Research Diets, New Brunswick, NJ) with or without sodium cholic acid (0.5% wt/wt) produced by mixing Purina mouse chow 5015 (750 g); casein, 80 mesh (75 g); dextrose (25 g); maltodextrin (42 g); sucrose (16.25 g); cellulose BW200 (12.5 g); cocoa butter (75 g); mineral mix S10001 (8.75 g); vitamin mix V10001 (2.5 g); choline bitartrate (1.25 g); and cholesterol USP (12.5 g).

Moderate uremia was induced by a two-step surgical procedure (3). Briefly, the upper and lower poles of the right kidney were resected in 10-wk-old mice. Two weeks later, the left kidney was removed. Control mice underwent sham operations at both 10 and 12 wk of age. Anesthesia was achieved with a mixture (ratio 3:0.2:4) of fentanyl (0.050 mg/ml), midazolam (5.0 mg/ml), and droperidol (2.5 mg/ml, 20 µl/g body wt sc), and buprenorphine 0.3 mg/ml (0.1 µg/g body wt sc twice daily for 3 days) was used as an analgesic after the surgical procedures. In mice on the C57BL76J background, NX does affect blood pressure and as such does not inflict insufficient adrenal function (3). NX and sham-operated mice were allocated to a cholesterol- and fat-enriched diet either with or without cholic acid. The NX mice fed the cholic acid-containing diet did not thrive and were killed before the end of the study. The cause of the poor compliance to the cholic acid-containing diet is unknown. At the time of death, several of the mice had grossly enlarged livers, gallstones, and elevated plasma liver enzyme concentrations.

Plasma biochemistry. Blood from the retroorbital venous plexus was collected in heparinized microtubes (Capiject, Terumo Medical, Elkton, MD) and centrifuged at 2,000 gfor 10 min at 4°C. Plasma was stored at –80°C. Plasma sodium, potassium, chloride, creatinine, total calcium, phosphate, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, albumin, and bilirubin concentrations were measured with a Modular P Hitachi Automatic Analyzer (Roche, Hvidovre, Denmark) using reagents from Roche. Plasma urea was measured with the same system or a Vitros 250 with reagents from the manufacturer (Ortho-Clinical Diagnostics, Rochester, NY). Cholesterol was measured with Modular P (plasma) or manually (gel filtration fractions and isolated HDL) using CHOD-PAP reagent (Roche). Phospholipids were measured by an enzymatic colorimetric method (Wako Chemicals, Neuss, Germany) and triglyceride by the GPO-Trinder method (Sigma Diagnostics, St. Louis, MO). Plasma SAA, soluble ICAM-1 (sICAM-1), and soluble VCAM-1 (sVCAM-1) were measured with monoclonal antibody-based sandwich enzyme-linked immunosorbant assays (Biosource, Nivelles, Belgium or R&D Systems Europa, Abingdon, Oxon, UK).

Gel filtration and ultracentrifugation of plasma. For assessing plasma lipoproteins, pooled plasma samples (200 µl) from 7–11 mice were separated by gel filtration chromatography at 20–24°C with PBS with Na2EDTA, pH 7.4 (PBS-EDTA; 0.1 g/ml) on serially connected Superose 6 and Superose 12 10/300 GL FPLC columns (Amersham Biosciences Europe, Horsholm, Denmark). The flow rate was 0.4 ml/min, and fractions of 250 µl were collected. Determination of V0 and Vt were done with intralipid and glycerol, respectively.

Plasma (stored at –80°C) from five, seven, and eight C57BL/6J NX mice (12 wk after NX) and three, four, and four sham-operated C57BL/6J mice used in another study (5) was used to make six plasma pools. HDL was isolated from each pool by sequential ultracentrifugation with a Beckman Ti 50.3 rotor and a Beckman Optima LE-80K ultracentrifuge (Beckman Coulter, Fullerton, CA). The density was adjusted with NaBr, and ultracentrifugation was performed at 100 000 rpm at 10°C for 5 h (d = 1.063 g/l) or 22 h (d = 1.21 g/l). The purified HDL (1.063 < d < 1.21 g/l) was dialyzed against PBS-EDTA at 4°C and stored at –80°C before analyses of cholesterol, phospholipids, and triglycerides.

Western blotting. Proteins were separated on 12% polyacrylamide gels (NuPAGE, Bis-Tris, Invitrogen, Tastrup, Denmark) and transferred to Hybond-P 0.45-µm polyvinylidene difluoride membranes (Amersham Biosciences) using a semidry electroblotter (Kem- En-Tec, Copenhagen, Denmark). The membranes were blocked for 1 h in skim milk containing wash buffer (50 mg/ml) followed by incubation for 16 h at 4°C with primary antibodies in the same solution (rabbit anti-mouse apoA-I, 1:5,000, Biosite, Taby, Sweden) or rabbit anti-mouse SAA (1:10,000, a kind gift from Dr. G. S. Getz, University of Chicago). After washing of the membranes, they were incubated with horseradish peroxidase-coupled goat anti-rabbit IgG antibodies (1:2,000, Dako, Ballerup, Denmark), washed again, and finally incubated for 3–4 min with SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemicals, Copenhagen, Denmark). Bands were detected in a chemiluminescence reader (FujiFilm LAS-1000 Intelligent Dark Box II, Fujifilm, Trorod, Denmark). Band intensities were quantified with the computer software Image Gauge ver. 4.0 (FujiFilm). For semiquantitative analyses of plasma apoA-I, we loaded 0.16 µl plasma/lane. Analyses of plasma dilutions with loadings of 0.0625, 0.125, and 0.25 µl showed proportionality between band intensities and amounts of loaded apoA-I.

mRNA quantification. After homogenization of mouse liver biopsies with a Tissuelyzer (Qiagen Retsch, Hann, Germany), total RNA was isolated with TRIzol (Invitrogen). Aortic RNA was made as previously described (5). RNA concentrations were assessed from the absorbance at 260 nm, and RNA integrity was ensured with RNA Nano LabChip (Agilent Technologies, Narum, Denmark). cDNA was made from 1 µg of total RNA with M-MULV reverse transcriptase (20 U, Roche) and random hexamer primers in 10-µl reactions at 37°C for 60 min. Real-time PCR with a LightCycler (Roche) was used to determine the mRNA expression of ICAM-1, VCAM-1, hypoxanthine phosphoribosyl transferase (HPRT), beta-actin, microsomal triglyceride transfer protein (MTP), apoB, apoA-1, and GAPDH. The forward and reverse primers were 5'-GTGGCTCTGGTCTTCCTGAC-3' (apoA-I-51) and 5'-ACGGTTGAACCCAGAGTGTC-3' (mapoA-I-31), respectively, for apoA-1 and 5'-TTGCGCTCATCTTAGGCTTT-3' (m-HPRT-31) and 5'-AAGCTTGCTGGTGAAAAGGA-3'(m-HPRT-51), respectively, for HPRT. Other primers have been described previously (2, 6, 21, 23). Each reaction mixture (20 µl) contained cDNA synthesized from 20 ng total RNA, 2.0–3.5 mM MgCl2, 10 pmol of each primer, 2 µl of LightCycler Faststart DNAmaster SYBR Green 1 mix (Roche), and PCR-grade water. Standard curves were made by serial dilutions of pools of mouse liver or aortic cDNA. All mRNAs were quantified in duplicate in separate runs. Agarose gel electrophoresis and DNA sequencing confirmed the specificity of the individual PCR reactions.

In the livers, the crude expression of beta-actin was lower in NX mice and cholic acid-fed mice than in sham-operated control (data not shown), whereas the crude expression of GAPDH was similar in the three groups. Hence GAPDH expression was used to normalize hepatic gene expression. In aortas, the expression of beta-actin, GAPDH, and HPRT was similar in NX and sham-operated mice, and the average expression of the three genes was used to normalize aortic gene expression. Importantly, the overall results and conclusions were similar whether the results were analyzed with or without normalization with housekeeping gene expression.

Evaluation of aortic pathology and quantification of atherosclerosis. To study aortic atherosclerosis, mice were anesthetized and the circulation was perfused with 0.9% NaCl (0°C) through a cannula placed in the left ventricle. The thoracic aorta was removed, freed of adventitial tissue, opened longitudinally, and the intimal surface was photographed with a digital camera. The heart with 1–2 mm of the aortic root was placed in OCT compound (Tissue-Tek, Sakura Fineteck, Varlose, Denmark) and frozen on dry ice. Before sectioning, hearts were fixed in phosphate-buffered paraformaldehyde (4% wt/vol, pH 7.0, Sigma-Aldrich, Vallensbak Strand, Denmark) and reembedded in OCT compound. Serial 10-µm-thick sections of the aortic sinus were cut in a cryostat. With the appearance of the first aortic valve, sections were collected on SuperFrost slides (Menzel-Glaser) precoated with 1% gelatin (Sigma). When all three valves were visible, 15 sections were collected on 3 slides with 5 sections on each. Two of the slides with sections of the best quality were stained with oil red O (Sigma) and hematoxylin (Sigma). Digital images were collected from six random sections at x4.0 magnification using a Nikon eclipse TE300 light microscope, a Nikon DXM 1200 digital camera, and the software control manager ACT-1. Oil red O-stained areas in the intima and inner media were measured with Leica IM50 Image Manager, version 4.0. The oil red O-stained area was expressed as the mean area in the six sections.

For staining of calcifications ad modum von Kossa, sections were incubated with 5% silver nitrate (Merck, Darmstadt, Germany) for 30 min in daylight, followed by 5% sodium thiosulfate (Sigma) for 3 min, and Nuclear Fast Red solution (0.1 g Nuclear Fast Red and 5 g aluminum sulfate hydrate in 100 ml distilled water) for 3 min. Between each staining, slides were washed in distilled water for 2–3 min. Sections from a calcified human atherosclerotic plaque were used as a positive control. von Kossa-stained sections were examined with a Leica S6E microscope.

For nitrotyrosine staining, OCT compound was removed by washing with TBS (0.05 M Tris, 0.15 M NaCl, and 0.01% Triton X-100, pH 7.6) for 2 x 5 min before treatment with 1 mM citric acid, pH 6.0, at 100°C for 15 min, and 1% H2O2 for 10 min. After being washed with distilled water, sections were incubated at room temperature with 5% goat serum (Dako) in TBS for 30 min and incubated for 1 h at room temperature with an antiserum directed against nitrotyrosine (1:500, Upstate) (3). After being washed three times for 5 min in TBS, bound antibodies were visualized with the EnVision+ System/HRP, rabbit (DAB+, Dako). Finally, after being washed in distilled water, sections were dehydrated through a series of alcohol solutions, and a coverglass was mounted with Pertex (HistoLab Products, Göteborg, Sweden). Sections incubated with rabbit immunoglobulins (Negative Control Rabbit Immunoglobulin Fraction, Normal, Dako) in the same immunoglobulin concentration as the primary antibodies were included as negative controls.

Statistical analyses. Statistical calculations were performed using GraphPad Prism, version 4.03 for Windows. Results were analyzed by Student's unpaired t-tests with Welch's correction when appropriate except for plasma SAA values, where the Mann-Whitney U-test was used. Data are presented as means ± SE, with n indicating the number of mice studied.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of NX and a cholic acid-containing diet on plasma markers of kidney and liver function. Plasma urea was increased 2.5- to 4.5-fold by NX and was unaffected by the cholic acid-containing diet (Fig. 1). Also, the plasma potassium, creatinine, and calcium concentrations were increased in the NX compared with the sham group, whereas the cholic acid-containing diet increased plasma calcium and decreased plasma phosphate (Table 1). NX did not affect plasma sodium and chloride concentrations (Table 1). Both NX and the cholic acid-fed mice gained less weight than the sham-operated controls (Table 1). Plasma alkaline phosphatase and lactate dehydrogenase were both increased 1.8-fold, whereas plasma alanine aminotransferase and albumin were decreased by 60 and 10%, respectively, in the NX group compared with the sham group (Table 2). The cholic acid-containing diet increased plasma alkaline phosphatase, lactate dehydrogenase, and alanine aminotransferase 2.3-, 3.8-, and 5.3-fold, respectively, compared with the sham group, but did not affect plasma bilirubin or albumin (Table 2).


Figure 1
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  		Fig. 1. Effect of nephrectomy (NX) and a cholic acid-containing diet on plasma urea in C57BL/6J mice. bullet, NX mice (n = 7); {circ}, sham mice (n = 11); {square}, cholic acid-fed mice (n = 7). Values are means ± SE. **P < 0.01 and ***P < 0.001 compared with sham mice.

 

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  		Table 1. Effect of NX and a cholic acid-containing diet on plasma creatinine, electrolytes, phosphate, and body weight

 

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  		Table 2. Effect of NX and a cholic acid-containing diet on plasma biochemical parameters related to liver function

 
Effect of NX and a cholic acid-containing diet on plasma lipoproteins and hepatic expression of MTP and apoB mRNA. NX did not affect the total plasma cholesterol concentration, whereas it was increased by the cholic acid-containing diet (Fig. 2A). On gel filtration analyses of pooled plasma from 4, 13, 19, and 26 wk after NX, there was a consistent, albeit small, elevation of VLDL/LDL cholesterol and a corresponding decrease in HDL cholesterol in the NX group compared with the sham group (Fig. 2B). The cholic acid-containing diet caused a massive increase in VLDL/LDL cholesterol (Fig. 2B).


Figure 2
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  		Fig. 2. Effect of NX and a cholic acid-containing diet on plasma cholesterol and lipoprotein distribution in C57BL/6J mice. A: total plasma cholesterol in NX (bullet, n = 7), sham ({circ}, n = 11), and cholic acid-fed mice ({square}, n = 7). Values are means ± SE. *P < 0.05 and ***P < 0.001 compared with sham mice. B: pooled plasma (200 µl) from 7–11 mice at 26 wk after NX was fractionated by gel filtration chromatography and cholesterol was measured in collected fractions. Solid line, NX mice (n = 7); long dashed line, sham mice (n = 11); short dashed line, cholic acid-fed mice (n = 7).

 
The hepatic production of VLDL is affected by the mRNA expression of both MTP and apoB (2, 23). The expression of liver MTP mRNA was reduced both by NX and by the cholic acid-containing diet (Fig. 3A), whereas the expression of apoB mRNA was decreased by the cholic acid-containing diet but not by NX (Fig. 3B).


Figure 3
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  		Fig. 3. Effect of NX and cholic acid-containing diet on liver expression of microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) mRNA. MTP (A) and apoB (B) mRNA expression levels were normalized with the expression of GAPDH mRNA in the same sample. Filled bars, NX mice (n = 7); hatched bars, cholic acid-fed mice (n = 6); open bars, sham mice (n = 10). Values are means ± SE. Brackets and P values show results of 2-group comparisons.

 
Effect of NX on HDL composition and serum amyloid A. To analyze the impact of NX on HDL lipid composition, we isolated HDL from NX and sham mice by sequential ultracentrifugation. The relative cholesterol content of HDL was significantly reduced in NX compared with sham mice, whereas the triglyceride content was increased and the phospholipid and total protein contents were unaffected (Table 3). The average plasma apoA-I concentration and hepatic apoA-I mRNA expression did not differ in a statistically significant manner between NX and sham mice (data not shown).


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  		Table 3. Lipid composition of HDL from NX and sham mice

 
The mean plasma SAA concentration increased markedly in the NX compared with the sham-operated mice (Fig. 4A). The cholic acid-containing diet also increased plasma SAA but to a lesser extent than NX (Fig. 4A). On gel filtration and Western blotting analyses of plasma from NX mice, SAA and apoA-I were associated with HDL-sized lipoproteins (Fig. 4B). It was striking that the magnitude of the increase in plasma SAA concentrations above the average value in sham-operated mice varied greatly between NX mice (~0- to 53-fold). The cause of this variation is unknown. The mice were kept in a clean facility with frequent testing excluding infections, none of the mice had signs of wound infection after surgery and the rise in SAA was not associated with the extent of uremia (judged from plasma urea concentrations). To examine whether an increase in plasma SAA was associated with a decrease in plasma apoA-I, we compared the plasma apoA-I concentration in individual NX mice with the plasma SAA concentration. NX mice with high-plasma SAA had low apoA-I concentrations (Fig. 4C). However, the hepatic apoA-I mRNA expression was not decreased in parallel with the increase in SAA (Fig. 4C).


Figure 4
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  		Fig. 4. Effect of NX and a cholic acid-containing diet on plasma serum amyloid A (SAA), lipoprotein association of SAA, and plasma apoA-1 in C57BL/6J mice. A: plasma SAA in NX (bullet), cholic acid-fed mice ({square}), and sham mice ({circ}). Values are means ± SE. *P < 0.05 compared with sham mice. B: Western blots with antibodies against apoA-I and SAA on pooled gel filtration fractions. NON-LP designates a pool of the fractions eluting after HDL on the gel filtration column (see Fig 2B). C: apoA-I Western blotting, intensity of apoA-I bands, plasma SAA concentrations, and liver apoA-I mRNA expression (normalized with GAPDH mRNA) in individual NX and sham mice.

 
Effect of NX on aortic expression of adhesion molecules. Vascular inflammation is a hallmark of the accelerated atherosclerosis occurring in hypercholesterolemic, uremic, apoE-deficient mice (5, 6). To examine whether moderate uremiamay have a similar effect in wild-type mice without overt hypercholesterolemia, we measured markers of aortic inflammation and oxidative stress. The aortic expression of VCAM-1 and ICAM-1 mRNA did not differ between in NX and sham-operated control mice when measured 12 wk after NX using aortic RNA from a previous study (Fig. 5A) (5). Also, the plasma concentrations of sICAM-1 and sVCAM-1 were similar in NX and sham-operated controls (Fig. 5B).


Figure 5
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  		Fig. 5. Effect of NX on aortic expression of ICAM-1 and VCAM-1 mRNA and plasma sICAM-1 and sVCAM-1 in C57BL/6J mice. A: aortic expression of ICAM-1 and VCAM-1 mRNA [normalized with hypoxanthine phospho ribosyl transferase (HPRT), beta-actin, and GAPDH as described in MATERIALS AND METHODS] 12 wk after NX. The aortas were harvested from NX and sham-operated control mice used in a previous study (5). Filled bars, NX mice (n = 18); open bars, sham mice (n = 11). B: plasma sICAM-1 and sVCAM-1 concentrations 26 wk after NX. Filled bars, NX mice (n = 7); hatched bars, cholic acid-fed mice (n = 6); open bars, sham mice (n = 9). Values are means ± SE. There were no statistically significant differences between NX and sham-operated mice for any of the variables depicted.

 
Effect of NX and a cholic acid-containing diet on aortic atherosclerosis and aortic inflammation. Twenty-six weeks after NX, there were no macroscopically visible atherosclerotic plaques in the thoracic aorta in either NX, cholic acid-fed, or sham-operated control mice (data not shown). On analyses of cross sections of the aorta proximally to the aortic valves, the oil red O-stained area was similar in NX and sham-operated control mice, whereas it was increased ~2-fold by the cholic acid-containing diet (Fig. 6A).


Figure 6
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  		Fig. 6. Effect of NX and a cholic acid-containing diet on aortic atherosclerosis and nitrotyrosine staining in C57BL/6J mice. A: area of oil red O-stained lesions 26 wk after NX. Filled bars, NX mice (n = 7); hatched bars, cholic acid-fed mice (n = 6); open bars, sham mice (n = 11). Values are means ± SE. Brackets and P values show results of 2-group comparisons. B–D: immunohistochemical nitrotyrosine staining (brown) and negative control staining (insets) of aortas from a C57BL/6J NX mouse (B), a C57BL/6J sham-operated control mouse (C), and an apoE-deficient NX mouse with atherosclerosis (D; the aorta was harvested 36 wk after NX and also used in a another study) (4). Note the uniform staining of nitrotyrosine in the media of NX and sham C57BL/6J mice as opposed to the strong nitrotyrosine staining of the foam cell intimal lesion in the apoE-deficient NX mouse.

 
NX apoE-deficient mice display increased accumulation of nitrotyrosine-stained material in the aorta, suggesting that oxidative stress may play an integrated role in uremic atherosclerosis (3). To examine aortic oxidative stress in wild-type NX mice, aortic sections were stained with an anti-nitrotyrosine antibody. Nitrotyrosine-stained material in NX and sham-operated control mice was uniformly distributed in the aortic media (Fig. 6, B and C). In contrast, in atherosclerotic aortas from uremic apoE-deficient mice harvested 26 wk after NX (4), nitrotyrosine was predominantly in intimal atherosclerotic lesions rather than in the media (Fig. 6D). There was no difference in the nitrotyrosine-staining intensity between aortas from NX and sham-operated control mice as judged from blinded semiquantitative assessments of four to five sections per mouse by two independent persons (data not shown).

As in our previous studies of apoE-deficient mice (3–5), we did not see any calcifications in the aortic root of NX, cholic acid-fed, or sham mice on von Kossa staining of four to five aortic sections from each mouse (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study suggests that moderate uremia increases VLDL/LDL cholesterol in wild-type mice. Human individuals with uremia also have increased VLDL due to impaired metabolism in plasma, involving decreased lipoprotein lipase activity and lipoprotein clearance in the liver (13, 31). Although we did not assess plasma lipoprotein turnover, the present results in mice may similarly reflect impaired clearance of apoB-containing lipoproteins since the gene expression of MTP in the liver was slightly decreased and that of apoB unchanged in the moderately uremic compared with the control mice.

Although NX did not affect the plasma HDL cholesterol concentration, the composition of the HDL particles was changed. NX conferred a marked elevation of HDL-associated SAA in wild-type mice. SAA is mainly produced in the liver. Thus the data suggest that moderate uremia causes an inflammatory response in the mouse liver. The effect of NX on plasma SAA levels varied greatly between mice, and the NX mice with the highest SAA concentrations also had low plasma apoA-I concentrations. Despite that the hepatic apoA-I gene expression is decreased in uremic rats (32), the hepatic apoA-I mRNA expression was not reduced in the NX mice. This suggests that chronic inflammation in mice with moderate uremia confers a decrease in plasma apoA-I, which is not necessarily due to lowering of the hepatic apoA-I gene expression. HDL from uremic mice also contained more triglycerides and less cholesterol per microgram protein than control HDL. These changes in HDL lipid composition are similar to those described after induction of acute inflammation with an injection of croton oil in rabbits or with LPS in baboons (8). Nevertheless, in mice LPS-induced acute inflammation does not change the lipid composition of HDL (despite increasing plasma SAA), suggesting that, at least in mice, chronic inflammation associated with uremia affects HDL differently than acute inflammation perhaps due to reduced hepatic lipase activity (16) and/or plasma LCAT activity (33). Interestingly, stable isotope turnover studies support the notion that triglyceride-enrichment of HDL increases its catabolic rate (19).

NX dramatically accelerates the development of atherosclerosis in the aortas of apoE- deficient mice, which have elevated plasma VLDL/LDL cholesterol (3). In apoE-deficient mice, NX also increases aortic expression of the genes encoding ICAM-1 and VCAM-1 as well as plasma concentrations of sICAM-1 and sVCAM-1 (4, 6). The increase in ICAM-1 expression (and perhaps also VCAM-1) precedes lesion formation in NX apoE-deficient mice, suggesting that vascular inflammation may be an important precipitating factor in uremic atherosclerosis. In the present study, neither the aortic expression of VCAM-1 and ICAM-1 mRNA nor the plasma concentrations of sICAM-1 and sVCAM-1 were increased in NX wild-type mice. This supports the idea that vascular inflammation in uremic mice to a large extent is dependent on concomitant hypercholesterolemia. Similarly, when aortic oxidative stress was examined with nitrotyrosine staining, we saw no difference between NX and sham-operated control mice. Of note, in NX apoE-deficient mouse aortas nitrotyrosine staining was predominantly within the intimal lesions. These results further suggest that the proatherogenic effect of uremia in mouse arteries relies on hypercholesterolemia. Accordingly, there was no difference in atherosclerotic lesion areas in the aortic arch between wild-type NX and sham-operated control mice, and the average lesion area in NX mice was less than in the cholic-acid fed mice. Thus the small increase in VLDL/LDL cholesterol and the proinflammatory changes in HDL composition were insufficient to induce atherosclerosis in NX C57BL/6J mice. Notably, this result does not exclude that formation of proinflammatory HDL contributes to accelerated lesion formation in the setting of more markedly elevated VLDL/LDL cholesterol concentrations such as in apoE-deficient mice and possibly humans.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported by an institutional grant from Rigshospitalet, University of Copenhagen, The Danish National Research Council, The Danish Heart Foundation, and "Fru Ruth E König-Petersens Forskningsfond".


   ACKNOWLEDGMENTS
 
We thank Dr. G. S. Getz, University of Chicago, for SAA antibodies and Kirsten Bang, Karen Rasmussen, Hanne Rasmussen, Charlotte Wandel, Hanne Kjærgaard, and Kirsten Hansen for excellent technical assistance at various stages of the project.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. B. Nielsen, Dept. of Clinical Biochemistry, KB3011, Rigshospitalet, Univ. of Copenhagen, Blegdamsvej 9, DK-2100, Copenhagen, Denmark (e-mail: larsbo{at}rh.dk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Attman PO, Samuelsson O, Alaupovic P. Progression of renal failure: role of apolipoprotein B-containing lipoproteins. Kidney Int Suppl 63: S98–S101, 1997.[CrossRef][Medline]
  2. Bartels ED, Lauritsen M, Nielsen LB. Hepatic expression of microsomal triglyceride transfer protein and in vivo secretion of triglyceride-rich lipoproteins are increased in obese diabetic mice. Diabetes 51: 1233–1239, 2002.[Abstract/Free Full Text]
  3. Bro S, Bentzon JF, Falk E, Andersen CB, Olgaard K, Nielsen LB. Chronic renal failure accelerates atherogenesis in apolipoprotein E-deficient mice. J Am Soc Nephrol 14: 2466–2474, 2003.[Abstract/Free Full Text]
  4. Bro S, Binder CJ, Witztum JL, Olgaard K, Nielsen LB. Inhibition of the renin-angiotensin system abolishes the proatherogenic effect of uremia in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 27: 1080–1086, 2007.[Abstract/Free Full Text]
  5. Bro S, Borup R, Andersen CB, Moeller F, Olgaard K, Nielsen LB. Uremia-specific effects in the arterial media during development of uremic atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 26: 570–575, 2006.[Abstract/Free Full Text]
  6. Bro S, Moeller F, Andersen CB, Olgaard K, Nielsen LB. Increased expression of adhesion molecules in uremic atherosclerosis in apolipoprotein-E-deficient mice. J Am Soc Nephrol 15: 1495–1503, 2004.[Abstract/Free Full Text]
  7. Buzello M, Tornig J, Faulhaber J, Ehmke H, Ritz E, Amann K. The apolipoprotein e knockout mouse: a model documenting accelerated atherogenesis in uremia. J Am Soc Nephrol 14: 311–316, 2003.[Abstract/Free Full Text]
  8. Cabana VG, Lukens JR, Rice KS, Hawkins TJ, Getz GS. HDL content and composition in acute phase response in three species: triglyceride enrichment of HDL a factor in its decrease. J Lipid Res 37: 2662–2674, 1996.[Abstract]
  9. Cheung AK, Sarnak MJ, Yan G, Dwyer JT, Heyka RJ, Rocco MV, Teehan BP, Levey AS. Atherosclerotic cardiovascular disease risks in chronic hemodialysis patients. Kidney Int 58: 353–362, 2000.[CrossRef][Web of Science][Medline]
  10. Coetzee GA, Strachan AF, van der Westhuyzen DR, Hoppe HC, Jeenah MS, de Beer FC. Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition. J Biol Chem 261: 9644–9651, 1986.[Abstract/Free Full Text]
  11. Foley RN, Parfrey PS, Sarnak MJ. Clinical epidemiology of cardiovascular disease in chronic renal disease. Am J Kidney Dis 32: S112–S119, 1998.[Web of Science][Medline]
  12. Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY. Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. N Engl J Med 351: 1296–1305, 2004.[Abstract/Free Full Text]
  13. Goldberg A, Sherrard DJ, Brunzell JD. Adipose tissue lipoprotein lipase in chronic hemodialysis: role in plasma triglyceride metabolism. J Clin Endocrinol Metab 47: 1173–1182, 1978.[Abstract/Free Full Text]
  14. Husebekk A, Skogen B, Husby G. High-density lipoprotein has different binding capacity for different apoproteins. The amyloidogenic apoproteins are easier to displace from high-density lipoprotein. Scand J Immunol 28: 653–658, 1988.[CrossRef][Web of Science][Medline]
  15. Ivanovski O, Szumilak D, Nguyen-Khoa T, Ruellan N, Phan O, Lacour B, scamps-Latscha B, Drueke TB, Massy ZA. The antioxidant N-acetylcysteine prevents accelerated atherosclerosis in uremic apolipoprotein E knockout mice. Kidney Int 67: 2288–2294, 2005.[CrossRef][Web of Science][Medline]
  16. Klin M, Smogorzewski M, Ni Z, Zhang G, Massry SG. Abnormalities in hepatic lipase in chronic renal failure: role of excess parathyroid hormone. J Clin Invest 97: 2167–2173, 1996.[Web of Science][Medline]
  17. Lewis KE, Kirk EA, McDonald TO, Wang S, Wight TN, O'Brien KD, Chait A. Increase in serum amyloid a evoked by dietary cholesterol is associated with increased atherosclerosis in mice. Circulation 110: 540–545, 2004.[Abstract/Free Full Text]
  18. Madore F. Uremia-related metabolic cardiac risk factors in chronic kidney disease. Semin Dial 16: 148–156, 2003.[CrossRef][Web of Science][Medline]
  19. Marsh JB, Welty FK, Schaefer EJ. Stable isotope turnover of apolipoproteins of high-density lipoproteins in humans. Curr Opin Lipidol 11: 261–266, 2000.[CrossRef][Web of Science][Medline]
  20. Massy ZA, Ivanovski O, Nguyen-Khoa T, Angulo J, Szumilak D, Mothu N, Phan O, Daudon M, Lacour B, Drueke TB, Muntzel MS. Uremia accelerates both atherosclerosis and arterial calcification in apolipoprotein E knockout mice. J Am Soc Nephrol 16: 109–116, 2005.[Abstract/Free Full Text]
  21. Moeller F, Nielsen LB. Aortic recruitment of blood lymphocytes is most pronounced in early stages of lesion formation in apolipoprotein-E-deficient mice. Atherosclerosis 168: 49–56, 2003.[CrossRef][Web of Science][Medline]
  22. Navab M, Ananthramaiah GM, Reddy ST, Van Lenten BJ, Ansell BJ, Hama S, Hough G, Bachini E, Grijalva VR, Wagner AC, Shaposhnik Z, Fogelman AM. The double jeopardy of HDL. Ann Med 37: 173–178, 2005.[CrossRef][Web of Science][Medline]
  23. Nielsen LB, Bartels ED, Bollano E. Overexpression of apolipoprotein B in the heart impedes cardiac triglyceride accumulation and development of cardiac dysfunction in diabetic mice. J Biol Chem 277: 27014–27020, 2002.[Abstract/Free Full Text]
  24. Nishina PM, Verstuyft J, Paigen B. Synthetic low and high fat diets for the study of atherosclerosis in the mouse. J Lipid Res 31: 859–869, 1990.[Abstract]
  25. O'Brien KD, McDonald TO, Kunjathoor V, Eng K, Knopp EA, Lewis K, Lopez R, Kirk EA, Chait A, Wight TN, de Beer FC, LeBoeuf RC. Serum amyloid A and lipoprotein retention in murine models of atherosclerosis. Arterioscler Thromb Vasc Biol 25: 785–790, 2005.[Abstract/Free Full Text]
  26. Qin P, Borges-Marcucci LA, Evans MJ, Harnish DC. Bile acid signaling through FXR induces intracellular adhesion molecule-1 expression in mouse liver and human hepatocytes. Am J Physiol Gastrointest Liver Physiol 289: G267–G273, 2005.[Abstract/Free Full Text]
  27. Schreyer SA, Wilson DL, LeBoeuf RC. C57BL/6 mice fed high fat diets as models for diabetes-accelerated atherosclerosis. Atherosclerosis 136: 17–24, 1998.[CrossRef][Web of Science][Medline]
  28. Song L, Schindler C. IL-6 and the acute phase response in murine atherosclerosis. Atherosclerosis 177: 43–51, 2004.[CrossRef][Web of Science][Medline]
  29. Stewart-Phillips JL, Gagnon RF, Lough J. Atherosclerosis in uremic mice. ASAIO Trans 35: 631–634, 1989.[Medline]
  30. Van Lenten BJ, Hama SY, de Beer FC, Stafforini DM, McIntyre TM, Prescott SM, La Du BN, Fogelman AM, Navab M. Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures. J Clin Invest 96: 2758–2767, 1995.[Web of Science][Medline]
  31. Vaziri ND. Dyslipidemia of chronic renal failure: the nature, mechanisms, and potential consequences. Am J Physiol Renal Physiol 290: F262–F272, 2006.[Abstract/Free Full Text]
  32. Vaziri ND, Deng G, Liang K. Hepatic HDL receptor, SR-B1 and Apo A-I expression in chronic renal failure. Nephrol Dial Transplant 14: 1462–1466, 1999.[Abstract/Free Full Text]
  33. Vaziri ND, Liang K, Parks JS. Down-regulation of hepatic lecithin:cholesterol acyltransferase gene expression in chronic renal failure. Kidney Int 59: 2192–2196, 2001.[Web of Science][Medline]
  34. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 258: 468–471, 1992.[Abstract/Free Full Text]




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