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NDT Advance Access published online on June 5, 2007
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfm168
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Renal protective effects of pitavastatin on spontaneously hypercholesterolaemic Imai Rats

Xiang-Ming Liang1, Haruhisa Otani2,4, Qin Zhou3, Yoshinori Tone4, Ryoichi Fujii4, Masatoshi Mune5, Susumu Yukawa4 and Tadao Akizawa6

1Department of Nephrology, Qilu Hospital of Shandong University, Shandong Province, China, 2Department of Nephrology and Blood Purification Medicine, Wakayama Medical University, Wakayama, Japan, 3Teaching Hospital of Shandong University of Traditional Chinese Medicine, Shandong Province, China, 4Ryoshukai Wakayama Kidney Disease Clinic, Wakayama, Japan, 5Department of Nutrition, Siebold University of Nagasaki, Nagasaki, Japan and 6Department of Nephrology, Showa University School of Medicine, Tokyo, Japan

Correspondence and offprint requests to: Haruhisa Otani MD PhD, Wakayama Kidney Disease Clinic, San Ei Building 5F, 5-1-8 Misono Cho, Wakayama City 640-8331, Japan. Email: hotani{at}wakayama-med.ac.jp



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. Independent of their lipid-lowering effects, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have renal protective effects on various models of progressive renal diseases, therefore, additional therapeutic advantages have been considered. In the present study, using spontaneously hypercholesterolaemic Imai rats, we examined the protective effects of pitavastatin on renal injuries and the oxidative modification of the low-density lipoprotein (LDL) and high-density lipoprotein (HDL), since oxidized lipoproteins are speculated to be involved in the mechanism of this rat strain's renal injuries.

Methods. Male Imai rats were treated with pitavastatin (n = 11) at a dose of 100 mg/kg diet or received no specific therapy as controls (n = 11) from 10 to 22 weeks of age. Body weight, urinary protein excretion and serum constituents were evaluated every 4 weeks. At the end of the study, the effects of pitavastatin on the susceptibility of serum LDL and HDL to oxidation, and renal histology were examined.

Results. Pitavastatin treatment did not affect hyperlipidaemia, but significantly reduced proteinuria and preserved creatinine clearance deterioration. At the end of the study, lag times for LDL and HDL oxidation were prolonged by the treatment of pitavastatin to 126 and 153%, respectively, compared with the controlled group. The glomerulosclerosis index (SI) for untreated controlled rats was significantly higher than that for the pitavastatin-treated group. An immunohistochemistry study showed significantly lower numbers of ED-1 positive macrophages in the glomeruli and interstitium in pitavastatin-treated rats compared with those controlled.

Conclusion. Pitavastatin treatment prevented renal injuries in Imai rats independent of lipid-lowering effects. Prevention of oxidative modification of LDL and HDL may play an important role on the beneficial effects of pitavastatin treatment.

Keywords: hyperlipidaemia; Imai rat; lipoprotein; oxidative modification; pitavastatin



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Recent experimental studies suggest that abnormalities in lipid metabolism frequently accompany renal diseases and may be important in the pathogenesis of progressive renal injuries [1–3]. The availability of reducing serum lipid has also been reported [4–6]. As a principal lipid-lowering agent, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) were well investigated in various animal models of progressive renal diseases, and it was found that these agents can ameliorate glomerulosclerosis and tubulointerstitial fibrosis and preserve renal function. In particular, beneficial effects can be achieved even in the absence of lipid alteration in some species [6–11]. Therefore, except for the direct lipid-lowering effect, additional therapeutic advantages such as anti-inflammatory, anti-fibrotic effects and endothelial cell protective actions were considered. [6,7].

Focal segmental glomerulosclerosis (FSGS) is seen in a variety of human glomerular diseases. In addition, several animal models of chronic non-immune-mediated glomerular diseases are characterized by the development of FSGS [12–14]. Although the important factors in the pathogenesis of FSGS are poorly understood, glomerular capillary pressure [13], coagulation factors [15] and abnormal lipid metabolism, etc. [1,14] are suspected to contribute to the development of FSGS. Spontaneously hypercholesterolaemic Imai rats (Imai rats) are considered to be a model of FSGS with nephrotic syndrome. This strain, originally derived from Sprague-Dawley rats, exhibits a spontaneous glomerulopathy that is marked by progressive FSGS, and enormously elevated urinary protein excretion rate, leading to a chronic renal insufficiency, especially in males. Onset of proteinuria is coupled with a progressive rise in plasma cholesterol and triglyceride concentration. Abnormal lipid metabolism is considered to participate in the mechanism of glomerular damage in this strain [14].

The oxidation of low-density lipoprotein (LDL) is believed to play an important role in the initiation of the progression of glomerulosclerosis, in a similar way as the atherosclerosis progression mechanism [16–22]. Additionally, high-density lipoprotein (HDL) is believed to protect LDL against oxidative modification, and may be impaired if HDL itself becomes oxidized [23–25].

In the present study, we examined the effects of pitavastatin, a new HMG-CoA reductase inhibitor, on the development of FSGS lesions in Imai rats. To test the antioxidative effect of pitavastatin, we also investigated whether pitavastatin can increase the resistance of LDL and HDL to copper-induced oxidation.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Experimental design
Imai rats, originally supplied by Takeda Pharmaceuticals Co. (Osaka, Japan), were bred by way of brother–sister mating and raised at the laboratory animal centre in Wakayama Medical University. Animal care was performed in accordance with the guidelines of Wakayama Medical University. Twenty-two male Imai rats at 10 weeks of age with a range of urinary protein excretion from 15 to 30 mg/day were randomly divided into two groups. Group 1 rats (Cont, n = 11) were fed with a laboratory diet (Funabashi farm, Chiba, Japan) as controls. The diet was based on AIN93G [26], and the protein content was increased to 40% with casein as the protein source to accelerate the progression of renal failure. Group 2 rats (PITA, n = 11) were fed on the same diet, but supplemented with pitavastatin at a dose of 100 mg/kg diet. Pitavastatin was provided by Kowa Co. (Nagoya, Japan). All the animals were housed in a climate-controlled space with 12 h day and night cycles and allowed free access to food and tap water.

Food intake was measured over a 3–6 day period at 12, 16 and 20 weeks of age. Body weight, blood pressure, urinary protein excretion, urinary micro-cholesterol excretion, fasting serum cholesterol, triglyceride, total protein, albumin, creatinine and blood urea nitrogen were all evaluated every 4 weeks from 10 to 22 weeks of age. With the final check, urinary 8-hydroxydeoxyguanosine (8-OHdG), serum HDL-cholesterol and lipid peroxides (LPO) were also evaluated. Then, the rats were sacrificed in deep anaesthesia with diethyl ether, and blood was collected from the hearts to determine the oxidative resistance of LDL and HDL when Cu2+ was used as the pro-oxidant. The kidneys were removed, blotted and weighed, and tissue from the left kidneys was fixed in 10% neutral buffered formalin for histology and 70% ethanol for immunohistochemistry, respectively.

Systolic blood pressure (SBP) was recorded in conscious rats with the tail-cuff method with a tail manometer–tachometer system (SR5000, Ueda, Japan), after we warmed the rats for l0 min at 37°C before measurement. To determine urinary protein and urinary micro-cholesterol excretion, rats were individually housed in metabolic cages for a 24 h fasting period, deprived of food but allowed free access to water, and then urine samples were collected. In addition, fasting blood samples were obtained via percutaneous puncture of the tail artery. Urinary protein, serum cholesterol, triglyceride, HDL-cholesterol, total protein, albumin, creatinine and blood urea nitrogen were determined with an autoanalyser (7170, Hitachi, Japan). Urinary micro-cholesterol excretion was determined by the ECC method [27,28]. Serum lipid peroxides were determined by the TBA method and creatinine clearance (Ccr) was corrected for 100 g body weight.

Urinary 8-OHdG excretion
We measured the urinary excretion of 8-OHdG as a marker of oxidative DNA damage. The 8-OHdG concentration in urine over 24 h was measured by enzyme-linked immunosorbent assay (ELISA) with a 8-OHdG ELISA kit (Japan Institute for the Control of Aging, Fukuroi, Japan) as we reported earlier [29]. The specificity of the monoclonal antibody N45.1 used in the competitive ELISA kit was previously established [30].

Oxidation of LDL and HDL
Since the Imai rats expressed marked hyperlipidaemia at 22 weeks of age, LDL and HDL were easily isolated. Five millilitres of blood from each rat was collected in polypropylene tubes containing EDTA (1 mg/ml final concentration) when the rats were sacrificed. The plasma was isolated by low-speed centrifugation and immediately processed for lipoprotein fractionation. LDL (d = 1.019–1.063) and HDL (d = 1.063–1.21) fractions were isolated by sequential ultracentrifugation at 120 000 rpm over 4 h at 4°C with a Hitachi CS 150GX ultracentrifuge with a S120AT2 rotor [31]. Before oxidation, LDL and HDL were rapidly filtrated through disposable desalting columns (Econo-Pac 10 DG, Bio-Rad) to remove EDTA. Then, the protein content of LDL and HDL was quantified with the modified Lowry method and diluted with PBS to a final concentration of 50 µg protein/ml. At time 0, Cu2SO4 (1 µmol/l final concentration) was added to LDL- and HDL-containing solutions, respectively, and incubated at 30°C in a UV-1600 spectrophotometer (Simadzu, Japan) equipped with a six-position thermostated cuvette holder. Changes in absorbance were continuously monitored spectrophotometrically at 234 nm every 2 min over 5 h to follow the formation of conjugated dienes. From the conjugated dienes absorbance curve, the lag time was determined as the indexes of LDL and HDL oxidation, as described by Esterbauer et al. [32].

Renal histological examination
Formalin-fixed kidney tissue was sectioned at 2 µm thickness for light microscopic study. The sections were stained with periodic acid-Schiff reagent (PAS stain) and periodic acid-methenamine silver (PAM stain). Histological evaluation was performed by a pathologist without knowledge of the origin of the samples. A semiquantitative score was used to evaluate the degree of glomerular sclerosis according to the method of Raij et al. [33]. At least 100 glomeruli were examined, and the severity of the lesion was graded from 0 to 4+, according to the percentage of glomerular involvement: a 1+ lesion represented an involvement of 25% of the glomerulus, while a 4 + lesion indicated that 100% of the glomeru1us was involved. An injury score (glomerulosclerosis index, SI) was then obtained by evaluating the percentage of the sclerotic area (0–4+) multiplied by the percentage of affected glomeruli. The extent of injury for each individual tissue specimen was obtained by adding the scores. In addition, tubulointerstitial damage (tubular dilatation, casts and interstitial fibrosis) was estimated by examining 10 cortical fields (100 x magnification), and semiquantitatively grading the degree of damage in each field at a 0–4+ scale. The mean of 10 randomly determined fields was calculated as an index of tubulointerstitial damage for each tissue specimen [3].

Immunoperoxidase staining for macrophages
Infiltration of macrophages was evaluated by immunoperoxidase staining with the EnVision system [EnVision kit/HRP (DAB), DAKO, Carpinteria, CA, USA] [34]. Briefly, ethanol-fixed kidney tissue was sectioned at 4 µm thickness, deparaffinized with xylene and rehydrated with graded ethanol. Endogenous peroxidase activity was inhibited by 10 min incubation with 0.03% H2O2. To reduce the background, non-specific binding was blocked by incubating with carrier protein in sodium azide (Protein block serum-free, DAKO, Carpinteria, CA, USA) for 20 min. Sections were first incubated with a monoclonal antibody against rat monocytes/macrophages (ED-1, Chemicon, Temecula, CA, USA) for 1 h at room temperature, and then incubated with polymeric conjugate for 1 h. Peroxidase activity was developed in 3, 3-diaminobendine (DAB). Mayer's haematoxylin was added as a counter stain.

ED-1-positive cells were randomly counted in 50 glomeruli and 10 tubulointerstitial fields (400 x magnification) per animal, but the glomeruli with global sclerosis were not included. The average number was used for the estimation, respectively.

Statistical analysis
All statistical computations were performed using StatView J-5.0, a commercial statistics program (SAS Institute Inc., Japan). The results are explained as arithmetic means ± SD. The distribution of the data at each time point was first analysed. Significant differences between the two groups were examined by the unpaired Student's t-test if the data was within normal distribution ranges. For data that was not normally distributed, the differences between the groups were analysed by the Mann–Whitney U-test. Repeated measures analysis of variance (ANOVA) was used to compare continuous data, such as body weight and urinary protein excretion among others, between the two groups during the experiment. P < 0.05 was considered to be statistically significant.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Food intake and body weight
The food intake of the two groups (Figure 1A) was similar and no significant differences were found during the experimental period, thus no adjustments were made in their diet to maintain equal caloric and protein intake between groups. As seen in Figure lB, the body weights of the two groups were similar and differences did not reach statistical significance at any time point. Blood pressure was measured four times during the experimental period, but no significant differences were seen between the two groups (data not shown).


Figure 1
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  		Fig. 1. Food consumption (A) and body weights (B) in control (filled circles) and pitavastatin-treated (open circles) Imai rats. Values are mean ± SD. Both food consumption and body weights change with age in a similar way between the two groups, without significant difference at any time point.

 
Urinary protein and micro-cholesterol excretion
At 10 weeks of age, before the experiment began, urinary protein excretion was similar for both groups (Figure 2A). Thereafter, pitavastatin showed a marked proteinuria-reducing effect in Imai rats. At 14 weeks of age (4 weeks after the initiation of pitavastatin treatment), protein excretion became significantly less in the pitavastatin-treated group than in control rats and this significant difference persisted for the remainder of the experiment. At 22 weeks of age, the average value of protein excretion was 251 ± 40 mg/24 h in the pitavastatin-treated group and 320 ± 85 mg/24 h in the control group. Repeated measures ANOVA also indicated that there was a significant difference between the two groups during the experimental period (P < 0.01, ANOVA). Urinary micro-cholesterol excretion increased with age as with urinary protein excretion, which was significantly lower during the study period in the pitavastatin-treated group compared with the control group (P < 0.05, ANOVA). The results are shown in Figure 2B.


Figure 2
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  		Fig. 2. Proteinuria (A) and urinary micro-cholesterol excretion (B) in control (filled circles) and pitavastatin-treated (open circles) Imai rats. Values are mean ± SD. Urinary protein excretion increased markedly from 10 to 22 weeks. Urinary protein excretion of pitavastatin-treated rats showed a significant low value compared with controls from 14 weeks and through the end of the experiment (P < 0.01, ANOVA). Change of urinary micro-cholesterol excretion was similar to urinary protein excretion, which was significantly low during the study period in the pitavastatin-treated group compared with the control group (P < 0.05, ANOVA).

 
Fasting serum cholesterol, triglyceride and HDL-cholesterol
As shown in Figure 3A and B, levels of fasting serum cholesterol and triglyceride rose progressively in control and pitavastatin-treated rats from 10 weeks of age, and reached marked high values at 22 weeks (cholesterol: Cont, 394 ± 58 mg/dl; PITA, 381 ± 54 mg/dl; and triglyceride: Cont, 267 ± 73 mg/dl; PITA, 256 ± 64 mg/dl). There was no significant difference in cholesterol or triglyceride levels between the two groups during the procedure and at the end of the experiment.


Figure 3
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  		Fig. 3. Cholesterol (A) and triglycerides (B) in control (filled circles) and pitavastatin-treated (open circles) Imai rats. Values are mean ± SD. Fasting serum cholesterol and triglyceride rose progressively in both the control and pitavastatin-treated rats from 10 weeks of age, and reached a markedly high value at 22 weeks. There were no significant differences in cholesterol or triglyceride levels between the two groups both during the procedure and at the end of the experiment (ANOVA and unpaired Student's t-test).

 
At the end of the experiment, serum HDL-cholesterol did not show any significant difference between the two groups (Table 1).


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  		Table 1. Serum concentration of total protein, albumin, HDL-cholesterol, lipid peroxide and urinary 8-OHdG at the end of the experiment

 
Serum constituent
The rats in both the groups progressed to renal failure with age. However, increases in serum creatinine (Figure 4A) and blood urea nitrogen (Figure 4B) levels were significantly ameliorated in pitavastatin-treated rats compared with control rats from 14 weeks of age to the end of the experiment (P < 0.05, ANOVA, respectively). Ccr rapidly declined during the experimental period in the two groups (Figure 5), but the deterioration was significantly preserved by pitavastatin treatment (P < 0.05, ANOVA). At the end of the experiment, pitavastatin-treated rats had a significantly higher creatinine clearance level (0.28 ± 0.018 ml/min/100 gBW) compared with control rats (0.20 ± 0.024 ml/min/100 gBW). Serum total protein and albumin showed no significant difference between the two groups at any time point in the experiment.


Figure 4
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  		Fig. 4. Serum creatinine and serum blood urea nitrogen (BUN) in control (filled circles) and pitavastatin-treated (open circles) Imai rats. Values are mean ± SD. The rats in both groups progressed to renal failure with age. However, increases of serum creatinine and BUN were significantly attenuated in pitavastatin-treated rats compared with control rats from 14 weeks of age through the end of the experiment (P < 0.05, ANOVA, respectively).

 

Figure 5
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  		Fig. 5. Creatinine clearance in control (filled circles) and pitavastatin-treated (open circles) Imai rats. Values are mean ± SD. Creatinine clearance decreased rapidly during the experimental period in pitavastatin-treated rats and controls, but it was preserved significantly with the pitavastatin treatment (P < 0.05, ANOVA).

 
Oxidation of LDL and HDL, serum LPO and urinary 8-OHdG
The initial lag phases for Cu2+-induced conjugated diene formation in LDL and HDL were significantly prolonged in rats treated with pitavastatin at 22 weeks of the experiment compared with control rats (LDL: PITA, 117.4 ± 17.8 min; Cont, 93.0 ± 10.3 min, P < 0.01; and HDL: PITA, 58.8 ± 5.9 min; Cont, 38.4 ± 12.4 min, P < 0.01) (Figure 6).


Figure 6
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  		Fig. 6. Lag time in control (filled boxes) and pitavastatin-treated (open boxes) Imai rats. The initial lag phases for Cu2+-induced conjugated diene formation in LDL and HDL were significantly prolonged in the rats treated with pitavastatin at 22 weeks of the experiment compared with the control rats (*P < 0.01).

 
At the end of the experiment, the values of serum LPO and urinary 8-OHdG were lower in the rats treated with pitavastatin than the control group, but a statistical difference was not observed (P = 0.079, P = 0.083, respectively) (Table 1).

Renal histology
By macroscopic observation, the kidneys of the control rats looked swollen, pale, granular and bigger in size. The ambi-kidney weight was significantly elevated compared with that of pitavastatin-treated rats (7.4 ± 1.8 vs 5.0 ± 0.8 g/rat, P < 0.001) (Table 2).


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  		Table 2. Ambi-kidney weight, glomerulosclerosis index, tubulointerstitial injury and ED-1 positive cells in glomeruli and tubulointerstitial fields

 
By microscopic observation, histological abnormalities included segmental mesangial sclerosis with matrix expansion, obliteration of glomerular capillary lumens and adhesions between the glomerular tuft and Bowman's capsule. As shown in Figure 7, in untreated control rats, marked pathological changes were observed in most glomeruli showing segmental or global sclerosis. Hyaline deposition in the glomerular tufts or adhesion of the tufts to Bowman's capsule was frequently observed. Some foam cells were expressed in the glomeruli and urinary tubules. However, these changes were more moderate in pitavastatin-treated group. As shown in Table 2, SI was significantly higher in untreated control rats than in pitavastatin-treated rats (138 ± 27 vs 96 ± 22, P < 0.01). Untreated control rats showed widespread tubular dilation, casts and interstitial fibrosis compared with pitavastatin-treated rats (mean score 2.03 ± 1.09 vs 0.64 ± 0.30, P < 0.01) (Table 2). It should be noted that the casts in medulla and the tubular dilation in cortical were remarkable, and this may explain why the kidneys were swollen, paler and bigger in macroscopic observation in control rats.


Figure 7
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  		Fig. 7. Pathological changes in control (B, D, F) and pitavastatin-treated (A, C, E) Imai rats at the end of the experiment.

 
Infiltration of macrophages
As shown in Figure 8, compared with the controls, significantly less of ED-1-positive monocytes/macrophages were detected in the glomeruli (P < 0.01) and interstitium (P < 0.01) of pitavastatin-treated rats at the end of the experiment (Table 2). And the significant relation between ED-1 positive cells in glomeruli and SI was made clear by the regression analysis (P < 0.05).


Figure 8
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  		Fig. 8. ED-1 staining in the glomeruli and interstitium of control (B, D) and pitavastatin-treated (A, C) Imai rats at the end of the experiment. Significantly less ED-1 positive monocytes/macrophages were detected in the glomeruli and interstitium of pitavastatin-treated rats than controls at the end of the experiment.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
In the present study, we tested the influence of pitavastatin on renal insufficiency using spontaneously hypercholesterolaemic Imai rats. The pitavastatin treatment resulted in a reduction in proteinuria, preservation of creatinine clearance deterioration and amelioration of glomerular injury and tubulointerstitial fibrosis. In addition, pitavastatin treatment was able to increase the resistance of LDL and HDL to oxidation, and prevent the infiltration of macrophages into the glomeruli and interstitium. Remarkably, no alteration of serum lipid was observed through the experimental period. Our findings suggest that these renal protective effects are independent of the lipid-lowering effect. The past experiments on rats also showed that statins would not decrease serum lipid level in rats [8–11]. This is believed that when HMG-CoA reductase in rats is inhibited by statin, mRNA expression of HMG-CoA will increase.

In vitro, the statins were demonstrated to reduce mesangial cell expression, the production of monocyte chemoattractant protein-1 (MCP-1) [35] and macrophage-colony stimulating factor (M-CSF), as well as vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) [10,36]. Moreover, they can also inhibit the activation of transcription factor (nuclear factor –{kappa}B, NF-{kappa}B) [37], which plays a role in gene expression involved in mesangial cell inflammatory responses [8,38]. Statins also inhibit the proliferation of cultured mesangial cells [38,39], renal epithelial tubular cells [40] and vascular smooth muscle cells [41,42].

Recently, Park et al. [8] reported that statins ameliorate angiotensin II-induced renal injury by the reduction of macrophage infiltration, expression of adhesion molecules, and activation of nuclear transcription factors including NF-{kappa}B and activating protein-1 independent of blood pressure- and cholesterol-lowering effects. Moriyama et al. [9] reported that statins suppress the degree of immunostaining of Advanced Glycation End product (AGE) and tubulointerstitial fibrosis in chronic unilateral ureteral obstruction (UUO) kidneys. Usui et al. [10] suggested that these therapeutic benefits are mediated by the reduction of oxidative stress, and ICAM-1 expression in the early phase of diabetes nephropathy.

Kidney mesangial cells and vascular smooth muscle cells are closely related in terms of origin, microscopic anatomy, histochemistry and contractility. Recently, the progression of glomerulosclerosis in lipid-mediated renal injury and its similarity to atherosclerosis were described [16,17]. The oxidation of LDL is crucial for involvement in atherogenesis, and the role of oxidized LDL in the progression of glomerulosclerosis was also investigated [19–22]. Mesangial cells also have LDL receptor and scavenger receptors similar to vascular smooth muscle cells [22]. Takemura et al. [18] indicated that in human nephritic kidneys, glomerular epithelial and mesangial cells express both LDL receptors and scavenger receptors. The accumulation of apolipoproteins can occur independent of plasma lipid level, and may be associated with mesangial expansion and proteinuria. Keane et al. [20] demonstrated that low concentrations of LDL stimulate human mesangial cell proliferation, and the oxidative modification of LDL mediates the toxic effects of high LDL concentrations on human mesangial cells. Previously, we demonstrated that apolipoprotein B, the main component of LDL, is frequently stained in the glomerular mesangial area in human kidney biopsy specimens, and is related to the degree of proteinuria and renal function [19]. Lee and Kim [21] demonstrated that oxidized LDL is mainly present in the lesions of glomerulosclerosis and mesangial areas as well. Moreover, HDL, which is present in tissue fluid at a greater concentration than LDL, may protect LDL against oxidative modification [23–25]. If HDL was oxidized, its participation in reverse cholesterol transport and its possible protection of LDL may be impaired [43]. We hypothesized that pitavastatin could ameliorate the progression of renal injury in Imai rats by increasing the resistance of serum lipoprotein to oxidation, and therefore investigated the susceptibility of LDL and HDL to oxidative modification.

Usually, rats have a relatively small amount of lipoprotein, and therefore complete isolation of the lipoprotein fraction is difficult [31]. In the present study, plasma cholesterol and triglyceride concentrations in Imai rats progressed to marked high values, so we could easily isolate enough LDL and HDL from a few millilitres of plasma when the animals were sacrificed at 22 weeks of age. By administering pitavastatin, the lag times for Cu2+-induced oxidation of LDL and HDL were significantly prolonged. Oxidatively modified LDL is taken up in a receptor-mediated process by macrophages derived from circulating monocytes to form foam cells. Sano et al. [44] found that in diabetic rats with hyperglycaemia and hyperlipidaemia, multiorgan foam cells exhibited positive staining for anti-monocyte/macrophage antibody, and that in the cytoplasm of glomerular foam cells revealed positive staining for anti-rat apolipoprotein B antibody, so they suggested that the origin of foam cells can be attributed to lipid-laden macrophages.

In the present study, light microscopy revealed that these foam cells were prominent in the renal glomeruli and tubules of Imai rats, but they were depressed in the pitavastatin-treated groups. Less ED-1 positive cells in the glomeruli and interstitium of pitavastatin-treated rats were also observed by immunoperoxidase staining. In our past observation of Imai rats, we found that the infiltration of macrophages in glomeruli was more remarkable in the early phase of FSGS, which is similar to the recent findings by Le Berre et al. [45] from progression of FSGS in another rat strain, that before the onset of FSGS in Buffalo/Mna rats, at a non-proteinuric stage, the production of some macrophage-associated cytokines, especially TNF-{alpha}, was already very strong. So when we counted the number of ED-1 positive cells in glomeruli, the glomeruli with sclerosis were not included. These histological and immunohisochemical results show that pitavastatin may prevent macrophage infiltration by increasing the resistance of LDL and HDL to oxidation.

In the present study, we examined urinary cholesterol excretion to estimate the excretion of HDL. Before our study, Short et al. [46] had detected apolipoprotein A1 in the urine of nephrotic subjects, and they believe the main component of urinary cholesterol is HDL. Leaked HDL, possibly oxidized HDL, may cause the tubular injury, although the precise mechanism for this is unknown.

Our observations suggest that in pitavastatin-treated rats, increasing resistance of LDL and HDL to oxidation may play an important role in the amelioration of glomerulosclerosis and tubulointerstitial fibrosis.

In conclusion, pitavastatin showed renal beneficial protective effects independent of the lipid-lowering effect in Imai rats, which represent a model of FSGS with hypercholesterolaemia. The resistance of LDL and HDL to oxidative modification increased due to treatment with pitavastatin, and may contribute to these beneficial effects.



   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
The authors greatly appreciate Dr Osamu Hotta of Sendai Shakaihoken Hospital, for his determination of urinary micro-cholesterol.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Moorhead JF, Chan MK, El-Nahas M, Varghese Z. Lipid nephrotoxicity in chronic progressive glomerular and tubulo-interstitial disease. Lancet (1982) 2:1309–1311.[Web of Science][Medline]
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Received for publication: 6.12.05
Accepted in revised form: 5. 3.07


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