Mesangial expansion associated with glomerular endothelial cell activation and macrophage recruitment is developing in hyperlipidaemic apoE null mice

Patrick Bruneval, Jean Bariéty, Marie-France Bélair, Chantal Mandet, Didier Heudes and Antonino Nicoletti

INSERM U430, Broussais Hospital, Paris, France



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Lipids are involved in the onset and/or the progression of renal diseases. ApoE null mice are hyperlipidaemic and thus represent an experimental model for the study of the effect of severe hypercholesterolaemia on renal lesion development.

Methods. ApoE null mice were studied at 6 weeks of age fed a normal chow, after 20 weeks on a normal chow (mild hypercholesterolaemia), or a 0.15% cholesterol Western diet (WD; severe hypercholesterolaemia). Age- and diet-matched C57/B6 mice were used as controls. Glomerular structure was assessed by histology, electron microscopy and computerized morphometry. Glomerular macrophage recruitment and {alpha}-smooth-muscle actin, PCNA, VCAM-1 and MHC class II (I-Ab) expressions were assessed by immunohistochemistry.

Results. ApoE null mice fed the WD developed mesangial expansion characterized by an increase in mesangial area (P<0.05 vs C57BL/6 mice at 20 weeks). In apoE null mice, this was accompanied by a glomerular inflammatory process as demonstrated by (i) the presence of foam cells, (ii) macrophage recruitment, (iii) a higher expression of the I-Ab activation marker and (iv) endothelial-cell activation (VCAM-1 expression in 100% of glomeruli and electron microscopy showing cytoplasmic foldings protruding in the capillary lumina). This might explain why we also observed blood monocytes adhering to glomerular endothelial cells.

Conclusions. In apoE null mice, severe hyperlipidaemia leads to glomerular injury characterized by glomerular endothelial cell activation and macrophage recruitment.

Keywords: adhesion molecules; apoE null mice; glomerular endothelial cells; hyperlipidaemia; macrophages; mesangial expansion



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Apolipoprotein E gene inactivation (apoE null) in mice generated the most widely studied model of atherosclerosis, mimicking human atherosclerosis from early lesions to advanced fibro-fatty plaques in the aorta and its branches. Furthermore, it allows modulation of the progression and the extension of atherosclerosis via interventions on immune response [1], or via pharmacological [2] or diet interventions [3]. That latter clearly demonstrate that apoE null mice are a lipid-dependent atherosclerosis model in which serum cholesterol levels are 600 mg/dl with normal chow [3] and are dramatically increased on lipid-rich chow such as Western-type diet (21% fat with 0.15% cholesterol), ranging from 1000 to 4400 mg/dl with no age effect [3]. The human counterpart of apoE null mice is a very rare genetic condition of apolipoprotein E deficiency, included in type III hyperlipoproteinaemia [4].

Renal involvement has been described in a case of type III hyperlipoproteinaemia [5]. In addition, this exceptional condition, hyperlipidaemia might be involved in the onset and/or in the progression of renal diseases. In humans, dyslipidaemia is considered as a factor contributing to the worsening of renal function in patients with pre-existing nephropathies [68] as well as a predictive factor for the decline of creatinine clearance in the general population [9]. With hyperlipidaemic mice such as apoE null mice, we have now the possibility to analyse the effects of mild and severe hypercholesterolaemia on renal-lesion development. The focus of this study was on (i) the precise type of glomerular pathology, detecting especially mesangial expansion and foam cell accumulation and (ii) the cellular mechanisms involved, including the role of resident glomerular cells vs that of recruited macrophages (Mø) and activation of these cells.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Animals and diets
The mice used in this study were the progeny of apoE null mice that were initially developed by Piedrahita et al. [10] and backcrossed to C57BL/6 mice for six generations. They were produced in the Karolinska Institute (Stockholm, Sweden). After weaning at 4 weeks, male mice were fed normal chow until 6 weeks. They were then randomly divided in two groups fed either normal chow [5.0% fat with <0.05% cholesterol (Brood stock feed R3, B & K Universal AB, Sollentuna, Sweden)] or Western diet (WD) [21% fat with 0.15% cholesterol (AnalyCen, Linköping, Sweden)] for 20 weeks. For controls, wild-type C57BL/6 mice were fed the same diets for the same durations. Groups (n=5) were as follows: apoE null and C57BL/6 mice at 6 weeks of age (t0) all on ND, and after 20 weeks (t20) on ND or WD. The animals were killed in carbon dioxide and the kidneys were immediately removed. Transversal hilar sections were fixed in alcoholic Bouin's solution for histology and morphometric analysis or in 10% formalin for immunohistochemistry and embedded in paraffin. Polar sections were snap frozen in liquid nitrogen for frozen section immunohistochemistry. Small pieces of the renal cortex were fixed in 2.5% glutaraldehyde for transmission electron microscopy.

Histology
Sections (5 µm thick) were stained with Masson's trichrome for assessment of kidney pathology, especially interstitial and glomerular fibrosis and foam cells. The number of foam-cell-positive glomeruli was measured out of 100 glomeruli in each mouse kidney.

Morphometry
Further sections (5 µm thick) were stained with periodic acid–Schiff (PAS). Morphometry of glomeruli was performed using computerized image analysis as described previously [11]. Briefly, a Nachet NS 15 000 image analysis processor (Nachet-Microvision, Evry, France) was connected to a 3 CCD camera on a microscope working with a x40 objective (final calibration 0.2637 µm/pixel), and to a microcomputer for storage of the morphometric measurements and for driving the image analysis processor according to a personal program written in C language by D.H. Thirty random glomeruli were assessed in each mouse kidney for the following parameters: glomerular surface area, mesangial surface area and mean thickness of Bowman's capsule. For each mouse kidney, the data were the mean±SEM of the 30 measurements of each parameter.

Immunohistochemistry
Expressions of {alpha}-smooth-muscle actin, desmin, and proliferating cell nuclear antigen (PCNA), markers of mesangial cell activation, of podocyte activation, and of cell proliferation, respectively, were detected in formalin-fixed paraffin-embedded sections. Monoclonal antibody to {alpha}-smooth-muscle actin (clone 1A4; Neomarkers, Fremont, CA, USA) was used at a dilution of 1/200, and monoclonal antibody to desmin (clone D33; Dako, Trappes, France) was used at a dilution of 1/100. Both were revealed with avidin–alkaline phosphatase kit (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA) and Fast red substrate (Dako). Antigen retrieval for detection of PCNA was achieved in a microwave oven at 750 W, three times 5 min in 2x SSC buffer (300 mmol/l standard saline citrate buffer pH 6.2). Monoclonal antibody to PCNA (PC10; Dako) was used at a dilution of 1/200 and revealed with avidin–peroxidase kit (Vectastain ABC kit) and amino-ethyl-carbazol substrate (Dako).

Expressions of vascular cell adhesion molecule-1 (VCAM-1) and of major histocompatibility complex (MHC) class II (I-Ab), and detection of Mø were performed in acetone-fixed frozen sections. Monoclonal antibodies to VCAM-1 (clone 429; PharMingen, San Diego, CA, USA) and to MHC class II (I-Ab) (clone KH74; PharMingen) diluted at 1/100, and to Mø (clone F4/80; Serotec, Oxford, UK) diluted to 1/200 were revealed with avidin–alkaline phosphatase or peroxidase kits (Vectastain ABC kits) and Fast red or amino-ethyl-carbazol substrates.

Negative controls consisted of mouse kidney sections incubated with non-immune mouse serum instead of monoclonal antibodies and revealed as described above. To determine the intensity of microwave-oven treatment necessary to detect PCNA without background, formalin-fixed paraffin-embedded mouse gut and kidney tissue sections were tested. The proper intensity was obtained when nuclear PCNA labelling was restricted to the proliferative cells, i.e. the bottom of the Lieberkühn crypts and rare tubular epithelial cells.

Labelling intensity was measured in 100 glomeruli in each mouse kidney as follows: percentage of positive glomeruli for {alpha}-smooth-muscle actin, VCAM-1, and MHC class II (I-Ab), number of positive cells in glomeruli (positive cell density) for PCNA and Mø.

Transmission electron microscopy
Glutaraldehyde-fixed cortical samples of five mice of all the groups at t20 (apoE null and C57BL/6 mice on ND or WD) were studied using standard transmission electron microscopy technique and observed with a Zeiss EM 10 electron microscope.

Lipid measurements
Total serum cholesterol, HDL cholesterol and triglycerides were assessed with commercial kits (Boehringer Mannheim, Mannheim, Germany).

Statistics
For all the parameters, the different groups were compared using ANOVA analysis with age, diet and strain as factors, followed by non-parametric Fischer and Bonferroni tests. P<0.05 was considered as significant.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Pathology of the renal involvement in apoE null mice
Cholesterol levels. As expected, on a normal diet apoE null mice had increased levels of total cholesterol, HDL cholesterol and triglycerides as compared with C57Bl/6 mice (Table 1Go). WD induced an increase in total and HDL cholesterol in both C57Bl/6 wild-type and apoE null mice (Table 1Go), but hypercholesterolaemia was much more pronounced in apoE null mice.


View this table:
[in this window]
[in a new window]
 
Table 1.  Lipid values

 
Mesangial expansion. At standard histology, apoE null mice fed WD for 20 weeks had patent diffuse or focal mesangial expansion in some glomeruli (Figure 1AGo), whereas all the glomeruli were normal in the other groups at t0 and at t20. The expansion of the mesangium was corroborated by transmission electron microscopy. Indeed, transmission electron microscopy showed mild mesangial accumulation of micro-fibrillar and granular extracellular matrix material only in t20 WD apoE null mouse glomeruli (Figure 1BGo). We next performed morphometric analysis in order to quantify the process of mesangial expansion. Morphometric analysis revealed an increase in the mesangial surface area in t20 WD apoE null mice vs that of the t20 WD C57BL/6 mice (P<0.05; Figure 1CGo). The mesangial areas in the other groups were similar to that of the t20 WD C57BL/6 mice. The glomerular surface area and the mean thickness of Bowman's capsule did not change between the different t20 groups (data not shown), indicating that the mesangial surface area was the only morphometric glomerular parameter modified by severe hypercholesterolaemia. All the measured morphometric parameters increased with age from t0 to t20 (time effect P<0.01 for glomerular surface area, P<0.05 for mesangial area and P<0.0001 for the mean thickness of Bowman's capsule).



View larger version (109K):
[in this window]
[in a new window]
 
Fig. 1.  (A) Diffuse mesangial expansion in t20 WD apoE null mouse kidney. Note that glomerular capillary lumina (arrows) are patent, confirming the absence of tissue shrinkage in that tissue section highlighting the pattern of mesangial expansion. Paraffin section. Masson's trichrome; bar=15 µm. (B) Microfibrillar and granular extracellular matrix accumulation (arrows) within the mesangium. L, lumen of a glomerular capillary; Mo, adhering non-foamy macrophage. Transmission electron microscopy; bar=1.5 µm. (C) Morphometric analysis of the mesangial areas: the mesangium is increased in t20 WD apoE null mice vs T20 WD C57BL/6 mice (P<0.05). It increases with time (P<0.05). C57BL/6 mice (filled columns) and apoE null mice (open columns). Means±SEM. Strain effect, *, time effect, #, P<0.05.

 
Foam-cell accumulation. Foam cells accumulated in glomeruli of apoE null mice at t20 (Figure 2A and BGo), but not in t0 apoE null mice or in C57BL/6 mice fed WD. The number of foam-cell-positive glomeruli in apoE null mice dramatically increased with WD, reaching 9.7±1.2% in t20 WD apoE null mice vs 0.5±0.5% in t20 ND apoE null mice (P<0.0001, Figure 2CGo). Foam-cell-positive t20 WD apoE null glomeruli contained several or many foam cells. The histological alterations were confined to the glomeruli except in rare t20 WD apoE null mouse kidneys that showed scanty interstitial foam cells. In this group, at transmission electron microscopy, frequent foam cells were observed in the mesangium appearing as microvacuolar lipid-laden cells (Figure 2BGo) while none was observed in the other groups. In addition, non-foamy glomerular intracapillary monocytes were observed adherent to the endothelium in t20 WD apoE null mice (Figures 1BGo, 3B and CGo).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2.  (A) Several foamy macrophages (arrows) are present in the mesangium of a t20 WD apoE null mouse glomerulus. Paraffin section. Masson's trichrome; bar=10 µm. (B) Microvacuolar lipid-laden macrophages (arrows) in the mesangium in a t20 WD apoE null mouse glomerulus. Podocytes are normal. L, lumina of glomerular capillaries. Transmission electron microscopy; bar=5 µm. (C) Percentage of foam-cell-positive glomeruli: these increase with diet {dagger}{dagger}{dagger} (P<0.001) and mouse strain *** (P<0.001) effects.

 


View larger version (91K):
[in this window]
[in a new window]
 
Fig. 3.  (A) PCNA labelling (arrow) in a rare positive glomerulus containing a single proliferating cell in a t20 WD apoE null mouse glomerulus. Paraffin section. PCNA immunohistochemistry; bar=15 µm. (B) Adhesion of a blood monocyte (Mo) to glomerular capillary endothelial cells in a t20 WD apoE null mouse glomerulus. The blood monocyte is not loaded with lipids. The glomerular endothelium exhibits cytoplasmic expansions (asterisks) indicating activation. L, lumen; M, mesangial cell. Transmission electron microscopy; bar=1.5 µm. (C) Non-foamy blood monocytes (Mo) adhering to the activated glomerular capillary endothelium in a t20 WD apoE null mouse glomerulus. An infiltrated foamy macrophage (arrow) is also visible in the mesangium. L, lumen. Transmission electron microscopy; bar=1.5 µm. (D) Macrophage infiltration (arrows) within a glomerulus in a t20 WD apoE null mouse glomerulus. Frozen section. F4/80 immunohistochemistry; bar=15 µm. (E) Glomerular macrophage density: the macrophages are recruited within the glomeruli with strain, ** (P<0.05) and time ## (P<0.01) effects. Diet induces a tendency to increase macrophage density in apoE null mice.

 

Cellular mechanisms of the glomerular injury in apoE null mice
Cell types involved in the glomerular lesions: resident glomerular cells vs recruited blood monocytes. The mesangial expansion could be due to mesangial-cell hyperplasia, but transmission electron microscopy and histology failed to confirm this. Furthermore, PCNA immunohistochemistry did not show any change between groups in glomerular-cell proliferation, indicating that the mesangial cells did not proliferate (Figure 3AGo). Moreover, the mesangium was infiltrated by lipid-laden cells exhibiting a peripheral condensed chromatin and an abundant round-shaped cytoplasm, which are features of macrophage lineage cells (Figure 2BGo). These foam cells probably formed after extravasating through the glomerular endothelium, since the adherent blood monocytes were not preloaded with lipids (Figures 1BGo, 3B and CGo). In agreement with these ultrastructural findings, immunohistochemistry with F4/80 antibody pointed out at an increase in the glomerular density of Mø in apoE null mice at t20 as compared with age-matched C57BL/6 mice (Figure 3D and EGo, mouse strain effect P<0.05). Diet induced a tendency to increase the Mø density in apoE null mice, but this did not reach significance. Mø density increased with time from t0 to t20 in both mouse strains (Figure 3EGo, time effect P<0.01). Scanty cortical and medullary interstitial Mø were observed only in t20 apoE null mice. The podocytes and the parietal epithelial cells were normal at histology and transmission electron microscopy. Thus, these features indicate that the intraglomerular foamy Mø recruited from peripheral blood monocytes are involved in the glomerular lesions.

Glomerular-cell activation. The {alpha}-smooth-muscle actin expression level is usually used as an index of mesangial-cell activation. In this study, it did not change significantly with time or diet, and both mouse strains expressed similar levels, indicating that the mesangial cells were not activated (data not shown). Desmin expression, considered as an activation marker of podocytes, was observed in some vascular smooth-muscle cells but never in podocytes, indicating that podocytes were not activated (data not shown). Furthermore, podocytes were normal at electron microscopy (Figure 2BGo and data not shown). One of the most intriguing observations in this study was that the glomerular capillary endothelial cells exhibited an activated phenotype in the t20 WD apoE null mice. The ultrastructural study showed that the glomerular capillary endothelial cell cytoplasmic membrane had an irregular pattern of their luminal aspect with many cytoplasmic foldings protruding in the capillary lumen (Figures 3B, CGo and 4AGo). Once again, contrasting with the glomerular capillary endothelial cell changes, the interstitial capillary endothelial cells were normal, indicating that the endothelial activation was not a general feature induced by severe hypercholesterolaemia in all the renal vascular bed. Furthermore, blood monocytes were frequently adherent to the endothelium (Figures 1BGo, 3B and CGo). Such events were rarely observed in t20 ND apoE null mice and never detected in the other groups. Corroborating these results, we found that the glomerular capillary endothelial cells acquired a strong expression of VCAM-1 in t20 apoE null mice fed either ND or WD in 100% of the glomeruli at t20, whereas VCAM-1 expression was mostly undetectable in C57BL/6 mice fed either ND or WD and in t0 apoE null mice (Figure 4B and CGo). The intraglomerular macrophages were also activated by hypercholesterolaemia, since the expression of the MHC class II (I-Ab) was dramatically increased with time in apoE null mice, whereas it was low in the other groups (Figure 4D and EGo). Thus, in t20 apoE null mice, glomerular capillary endothelial cells and recruited intraglomerular Mø express activation markers.



View larger version (67K):
[in this window]
[in a new window]
 
Fig. 4.  (A) Activated capillary endothelial cell exhibiting cytoplasmic projections (asterisks) in the capillary lumen (L) in a t20 WD apoE null mouse glomerulus. Transmission electron microscopy; bar=1 µm. (B) Endothelial cell activation is evidenced by the VCAM-1 expression in the glomerulus (G) of a t20 WD apoE null mouse. Note the positivity of the adjacent arterial endothelium (asterisk) and of some interstitial capillaries (arrowheads). Frozen section. VCAM-1 immunohistochemistry; bar=15 µm. (C) Percentage of VCAM-1 positive glomeruli: VCAM-1 glomerular expression is increased with strain *** (P<0.0001) and time ### (P<0.0001) effects. (D) I-Ab-positive cells in a t20 WD apoE null mouse glomeruli (G). I-Ab-expressing cells are probably activated macrophages. Frozen section. I-Ab immunohistochemistry; bar=20 µm. (E) Percentage of I-Ab positive glomeruli. I-Ab expression is increased with strain *** (P<0.0001) and time ### (P<0.0001) effects.

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The main result of this study is that apoE null mice develop renal injury, the expression of which is full-blown on WD, inducing severe hypercholesterolaemia, and is characterized by diffuse mesangial expansion and foam cell accumulation. Its cellular pathophysiology involves Mø recruited from blood monocytes and glomerular capillary endothelial cells. Both cell types are activated, as reflected by foamy transformation and MHC class II (I-Ab) expression in Mø and by de novo endothelial VCAM-1 expression, adhesion of monocytes to the endothelium and luminal pseudopod formation in glomerular capillary endothelial cells.

Glomerulosclerosis can develop during diet-induced hypercholesterolaemia in normal [12,13], uninephrectomized [13] or type II diabetic [14] rats and during endogenous hyperlipidaemia in specific strains such as the Zucker rats [15]. We also found that hypercholesterolaemic apoE null mice develop patent mesangial expansion and exhibit accumulation of glomerular foam cells. They represent an interesting alternative rodent model of lipid glomerular injury, given the possibilities of diet or experimental interventions. Furthermore, the levels of hypercholesterolaemia reached in apoE null mice are much above those observed in diet-induced hypercholesterolaemic rats.

Among the resident glomerular cells, several cell types could be involved in the glomerular injury induced by hyperlipidaemia such as podocytes and mesangial cells. It has been shown that podocytes are damaged in uninephrectomized hyperlipidaemic rats, as revealed by ultrastructural changes and desmin expression [13]. In contrast, the podocytes were normal in our study, suggesting that the podocyte is not a target cell in cholesterol-induced glomerular injury.

Mesangial cells can be a target cell of hyperlipidaemia as well and may play a role in glomerular injury since they have a major contribution to the extracellular matrix production. Although mesangial expansion was present in t20 WD apoE null, our study did not show mesangial-cell activation as observed by {alpha}-smooth-muscle actin labelling, a well-established marker of mesangial cell activation. Furthermore, PCNA labelling did not detect any cell proliferation. This discrepancy between mesangial expansion and absence of mesangial cell activation has already been noted in rat models of hypercholesterolaemia and hypertriglyceridaemia [13]. Glomerulosclerosis triggered by immune injury is generally associated with mesangial activation, while glomerulosclerosis induced by chronic metabolic stress does not appear to be associated with detectable mesangial activation [13].

Another interesting feature of mesangial cells is their ability to bind lipoproteins via receptors [16]. Thus, these cells might contribute to lipid accumulation in the glomeruli. However, our study does not support this hypothesis. On the contrary, the intra-mesangial foam cells belonged to the Mø lineage and were not mesangial cells, as demonstrated by electron microscopy. One of the main observations in this study was the glomerular recruitment of Mø. Though this was previously described in hyperlipidaemic rats [12,13], we additionally showed that monocytes adhering to the activated glomerular endothelium of severe hypercholesterolaemic mice were not preloaded with lipids, indicating that foamy transformation occurred after extravasation from blood to the mesangial area. This strongly suggests that monocytes are recruited to the glomerulus because of lipid deposition in the mesangial compartment. In fact, it has been shown that Mø infiltrated in the mesangium in hyperlipidaemic rats co-localize with lipoproteins [17,18] and that the recruited Mø have the properties to transform into foam cells. Furthermore, oxidized lipoproteins enhance the inflammatory properties of Mø, resulting in the production of the glomerulosclerosis-promoting transforming growth factor-ß1 [12]. Therefore, once infiltrated into the glomeruli, the Mø probably contribute to the development of mild glomerular inflammation and of glomerulosclerosis, such as in diabetes [11].

The preferential homing of Mø to the glomeruli might be mediated by local activation of endothelial cells and glomerular capillary endothelial cell activation has been observed in the present study. This phenomenon might be related to the endothelial dysfunction reported in apoE null mice [19]. Our study thus suggests that the hypercholesterolaemia-associated endothelial dysfunction is not limited to the intima of large arteries but also spreads to the microcirculation. Interestingly, we also observed that the endothelial activation was not distributed uniformly within the kidney vasculature, but was largely prominent in the glomerular capillaries. In this compartment, hyperlipidaemia results in an increased adhesiveness of the endothelial cells with de novo expression of VCAM-1 and adhesion of monocytes. It will now be interesting to study which factors determine the differential susceptibility of these various endothelial compartments since increased endothelial adhesiveness is probably the key event for Mø recruitment in the glomerulus [11], as it is for Mø recruitment in the large arteries [20].

There were no statistically significant differences between apoE null mice and wild-type mice at t0 except for VCAM-1 expression, while plasma cholesterol levels in apoE null mice were already 2–3-fold higher than those in wild-type mice. This might indicate that hypercholesterolaemia per se is not sufficient to induce the effects reported in this study after 20 weeks of diet. Instead, age would rather exacerbate the hypercholesterolaemia-dependent alterations. This implies that chronic exposure to hypercholesterolaemia is required to observe the deleterious effects of high cholesterol levels on the kidney.

ApoE null mice are a model for the type III hyperlipoproteinaemia, which is a rare form of dyslipidaemia. While patients presenting with this disorder suffer from cardiovascular manifestations rather than renal lesions, alterations of the kidney have seldom been described. Thus, in such patients, glomerular lesions were detected that were characterized by foam-cell accumulation [5], quite similar to those described here in apoE null mice. Our data show for the first time that chronic, severe hypercholesterolaemia is also associated with glomerular capillary endothelial-cell activation, which in turn is likely to be involved in the recruitment of blood monocytes. Once infiltrated, these macrophages transform into foam cells, which may contribute to the development of the mesangial expansion observed in severe hypercholesterolaemic apoE null mice. Further studies will have to be performed with different apoE null mouse lines and different strains of hypercholesterolaemic mice in order to ascertain that the effects reported in the present study are not influenced by genetic factors in addition to hypercholesterolaemia.



   Acknowledgments
 
We thank Anh-Thu Gaston for excellent technical help.



   Notes
 
Correspondence and offprint requests to: Dr P. Bruneval, INSERM U430, Broussais Hospital, 96 rue Didot, 75014 Paris, France. Email: patrick.bruneval{at}hop.egp.ap-hop-paris.fr Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Nicoletti A, Caligiuri G, Hansson GK. Immunomodulation of atherosclerosis: myth and reality. J Int Med2000; 247:397–405[ISI][Medline]
  2. Knowles JW, Reddick RL, Jennette JC et al. Enhanced atherosclerosis and kidney dysfunction in eNOS-/-Apoe-/- mice are ameliorated by enalapril treatment. J Clin Invest2000; 105:451–458[Abstract/Free Full Text]
  3. Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb1994; 14:133–140[Abstract]
  4. Ghiselli G, Schaeffer EJ, Gascon P, Brewer HB. Type III hyperlipoproteinemia associated with apolipoprotein E deficiency. Science1981; 214:1239–1241[ISI][Medline]
  5. Amatruda JM, Margolis S, Hutchins GM. Type 3 hyperlipoproteinemia with mesangial foam cells in renal glomeruli. Arch Pathol1974; 98:51–54[ISI][Medline]
  6. Appel G. Lipid abnormalities in renal disease. Kidney Int1991; 39:169–183[ISI][Medline]
  7. Samuelson O, Mulec H, Knight-Gibson C et al. Lipoprotein abnormalities are associated with increased rate of progression of human chronic renal insufficiency. Nephrol Dial Transplant1997; 12:1908–1915[Abstract]
  8. Hunsickler LG, Adler S, Gaggiula A et al. Predictors of the progression of renal disease in the Modification of Diet in Renal Disease Study. Kidney Int1997; 51:1908–1919[ISI][Medline]
  9. Muntner P, Coresh J, Smith C, Eckfeldt J, Klag MJ. Plasma lipids and risk of developing renal dysfunction: The Atherosclerosis Risk in Communities Study. Kidney Int2000; 58:293–301[ISI][Medline]
  10. Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc Natl Acad Sci USA1992; 89:4471–4475[Abstract]
  11. Prigent-Sassy C, Heudes D, Mandet C et al. Early glomerular macrophage recruitment in streptozotocin-induced diabetic rats. Diabetes2000; 49:466–475[Abstract]
  12. Guijarro C, Kasiske BL, Kim Y et al. Early glomerular changes in rats with dietary-induced hypercholesterolemia. Am J Kidney Dis1995; 26:152–161[ISI][Medline]
  13. Joles JA, Kunter U, Janssen U et al. Early mechanisms of renal injury in hypercholesterolemic or hypertriglyceridemic rats. J Am Soc Nephrol2000; 11:669–683[Abstract/Free Full Text]
  14. Domiguez JH, Tang N, Xu W et al. Studies of renal injury III: lipid-induced nephropathy in type II diabetes. Kidney Int2000; 57:92–102[ISI][Medline]
  15. Lavaud S, Michel O, Sassy-Prigent C et al. Early influx of glomerular macrophages precedes glomerulosclerosis in the obese Zucker rat model. J Am Soc Nephrol1996; 7:2604–2615[Abstract]
  16. Wheeler DC, Fernando RL, Gillett MP et al. Characterisation of the binding of low-density lipoproteins to cultured rat mesangial cells. Nephrol Dial Transplant1991; 6:701–708[Abstract]
  17. van Goor H, van der Horst ML, Atmosoerodjo J et al. Renal apolipoproteins in nephrotic rats. Am J Pathol1993; 142:1804–1812[Abstract]
  18. Magil AB. Interstitial foam cells and oxidized lipoprotein in human glomerular disease. Mod Pathol1999; 12:33–40[ISI][Medline]
  19. Yang R, Powell-Braxton L, Ogaoawara AK et al. Hypertension and endothelial dysfunction in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol1999; 19:2762–2768[Abstract/Free Full Text]
  20. Nakashima Y, Raines EW, Plump AS, Breslow JL, Ross R. Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse. Arterioscler Thromb Vasc Biol1998; 18:842–851[Abstract/Free Full Text]
Received for publication: 12. 4.02
Accepted in revised form: 12. 9.02