Lipid-lowering-independent effects of simvastatin on the kidney in experimental hypercholesterolaemia

Stephanie H. Wilson2, Alejandro R. Chade1, Ariel Feldstein3, Tatsuya Sawamura4, Claudio Napoli5,6, Amir Lerman2 and Lilach O. Lerman1,

1 Division of Hypertension and 2 Division of Cardiovascular Diseases, Department of Internal Medicine and 3 Department of Pediatrics, Mayo Clinic, Rochester, MN, USA, 4 National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka, Japan, 5 Department of Medicine, University of Naples, Italy and 6 Department of Medicine-0682, University of California, San Diego, CA, USA



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Hypercholesterolaemia (HC), an independent risk factor for renal injury, is associated with formation of oxidized low-density-lipoprotein (ox-LDL), increased oxidative-stress and renal inflammation. HMG-CoA-reductase inhibitors are commonly used in HC, but their effects on renal haemodynamics and function in HC are poorly understood.

Methods. Pigs were studied after a 12-week normal diet, a 2% high-cholesterol diet (HC) or an HC diet supplemented with simvastatin (HC+simvastatin, 80 mg/day) (n=6–8 each group). Renal haemodynamics and function were quantified in vivo with electron-beam computed tomography (EBCT). Shock-frozen renal tissue was subsequently studied using immunohistochemistry.

Results. LDL cholesterol was similarly increased in HC and HC+simvastatin. Simvastatin-treated animals showed increased expression of endothelial nitric-oxide-synthase (eNOS), and decreased expression of the ox-LDL receptor LOX-1 in renal endothelial cells. Simvastatin also decreased tubular immunoreactivity of inducible-NOS, nitrotyrosine, nuclear-factor-{kappa}B, and tubuloglomerular trichrome staining. These were associated with a significant increase in cortical (6.1±0.1 vs 5.0±0.3 and 5.0±0.1 ml/min/cc, respectively, P<0.001) and medullary perfusion in HC+simvastatin compared to normal and HC.

Conclusions. Simvastatin attenuated the inflammatory and pro-oxidative environment as well as fibrosis in kidneys in pigs with diet-induced HC, in association with enhanced renal perfusion. These cholesterol-lowering-independent changes imply novel renoprotective effects of statins in the setting of HC and atherosclerosis.

Keywords: HMG-CoA reductase inhibitors; hypercholesterolaemia; kidney



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Lipid abnormalities often accompany and exacerbate renal disease [1,2], and hypercholesterolaemia (HC) is considered an independent risk factor for renal injury [3]. We have previously shown that even a short exposure to diet-induced HC is associated with an increase in oxidative stress, increased formation of oxidized low-density lipoprotein (ox-LDL), and renal inflammation [4,5], changes that may initially be counterbalanced by compensatory mechanisms, but can eventually compromise renal function.

Treatment of hyperlipidaemia with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) has led to a significant reduction in major cardiovascular events in humans [6,7]. Recent evidence suggests that even in the absence of cholesterol lowering, therapeutic benefits can be achieved in HC due to the direct tissue effects of statins on upregulation of endothelial nitric oxide (NO) synthase (eNOS) with increased bioavailability of NO, decreased cellular proliferation, and/or decreased oxidative stress [8,9]. Alterations in these pathways are often involved in renal disease progression, and in vitro and in vivo findings suggest that statins can provide protection against kidney diseases characterized by inflammation and/or enhanced proliferation of epithelial and mesangial cells [10]. However, there is a paucity of data regarding their effects on the kidney in HC, especially in conjunction with renal regional haemodynamics and function in vivo.

Electron-beam computed tomography (EBCT) is an ultra-fast scanner, which provides accurate, reproducible and non-invasive quantification of single kidney volume, perfusion and glomerular filtration rate (GFR) [11]. This technique can resolve subtle alterations in regional renal haemodynamics and function and may thus shed light on renal regional effects of statins. The present study was therefore designed to test the hypothesis that chronic treatment with the HMG-CoA reductase inhibitor simvastatin would ameliorate renal inflammation and fibrosis, improve immunoreactivity of NOS, and increase renal perfusion in HC, even in the absence of cholesterol lowering.



   Subjects and methods
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 Subjects and methods
 Results
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This study was approved by the Institutional Animal Care and Use Committee. Experiments were conducted on female domestic crossbred pigs (55–65 kg). One group of pigs (HC; n=6) was placed on a 12-week diet of 2% cholesterol and 15% lard by weight (TD 93296, Harlan Teklad, Madison, WI) [5]. Additional pigs were fed a similar diet with oral daily supplementation (80 mg/day) with the HMG-CoA reductase inhibitor simvastatin (HC+simvastatin; n=8; Merck). An additional group of six pigs fed a normal diet were used as controls.

After 12 weeks of diet, in vivo EBCT studies were performed for assessment of regional renal perfusion, renal blood flow (RBF), and glomerular filtration rate (GFR). A blood sample was obtained for measurement of sodium, potassium, creatinine (spectrophotometry), plasma lipid profile (Roche, Nutley, NJ), plasma renin activity (PRA; radioimmunoassay) as well as PGF2 alpha isoprostanes (EIA, Cayman), as a measure of oxidative stress [12].

Following completion of studies, the pigs were euthanized with i.v. (100 mg/kg) Sleepaway® (sodium pentobarbital, Fort Dodge Laboratories, Inc., Fort Dodge, IA). The kidneys were immediately dissected, and sections shock-frozen in liquid nitrogen (and maintained at -80°C) or preserved in formalin. In vitro studies were performed in either frozen or deparaffinized 5-µm-thick cross-sections, to evaluate immunoreactivity for eNOS, inducible-NOS (iNOS), nitrotyrosine (as a footprint for peroxynitrite formation in vivo), the proinflammatory nuclear-factor-{kappa}-B (NF{kappa}B), and the lectin-like specific receptor for ox-LDL, LOX-1. In addition, renal morphology and fibrosis were evaluated in 5-µm-thick cross-sections stained with H&E and trichrome.

Immunostaining for eNOS, nitrotyrosine and LOX-1
This was performed in frozen 5-µm-thick cross-sections, as described previously [5]. Monoclonal antibodies for eNOS (Transduction Laboratories, Lexington, KY; 1:500), nitrotyrosine residues (Cayman, Ann-Arbor, MI, 1:20) [5], or LOX-1 (1:280), served as primary antibodies. The Vectastain-Elite ABC Kit (Vector Laboratories, Burlingame, CA) was used, following the vendor's instructions.

Immunostaining for iNOS and NF{kappa}B
This was performed on deparaffinized renal 5-µm-thick cross-sections. Polyclonal (iNOS, Affinity Bioreagents, Golden, CO; 1:500) and monoclonal (NF{kappa}B, Santa Cruz Biotechnology Inc., CA; 1:50) primary antibodies were utilized. The secondary antibody, IgG Envision Plus (Dako), was followed by staining with the Vector NovaRED substrate kit (Vector-Laboratories, Burlingame, CA), and slides were counter-stained with haematoxylin [5].

Histology sections of the whole kidney were examined using a computer-aided image-analysis program (MetaMorph, Meta Imaging Series 4.6). In each representative slide, immunostaining was semi-automatically quantified in 15–20 fields by the computer program, and expressed as a percentage of staining of total surface area, and the results from all fields were averaged [5].

EBCT studies
On the day of the EBCT study, each animal was anaesthetized with intra-muscular ketamine (20 mg/kg) and xylazine (2 mg/kg), intubated, and mechanically ventilated with room air. Anaesthesia was maintained with a mixture of ketamine (15.7 mg/kg/h) and xylazine (2.3 mg/kg/h) in saline administered via an ear-vein cannula (0.05 ml/kg/min). This was followed by placement of intra-vascular catheters, as previously described [5]. Briefly, intra-arterial and i.v. catheters were placed at the suprarenal aorta and the right atrium for monitoring mean arterial pressure (MAP) and for administration of contrast media, respectively.

Animals were then transferred to the EBCT (Imatron C-150, Imatron Inc. South San Francisco, CA) scanning gantry. After a 1-h recovery period during which saline (5 ml/min) was administered, haemodynamic parameters were recorded and blood samples were collected. An EBCT study of renal perfusion and function was then performed over the mid-hilar section of both kidneys during respiratory suspension [5,11]. Forty consecutive scans (over 3 min) were obtained at variable time intervals after a bolus injection (0.5 cc/kg over 1 s) of the non-ionic, low-osmolar contrast medium iopamidol (Isovue® -370, Squibb Diagnostics, Princeton, NJ) into the right atrial catheter. Lastly, a renal volume study was performed for measurement of cortical and medullary volumes [12].

EBCT data analysis
The methodology used for EBCT data analysis has been previously described in detail [5,11]. Briefly, regions of interest were selected from the images by tracing the aorta and the bilateral renal cortex and medulla. Time-density curves were generated for each region, and fitted with extended gamma-variate functions to obtain the area and first moment (mean transit time, s) of each vascular curve [5,11]. Renal regional perfusion (ml/min/cc tissue), normalized single-kidney GFR (ml/min/cc tissue), cortical, medullary volumes, and RBF were subsequently calculated as previously described in detail [5,11].

Statistical analysis
Results are expressed as mean±SEM. Data were compiled from both kidneys. Statistical comparisons between experimental periods within groups were performed using paired Student's t-test, and among groups using analysis of variance (ANOVA), with the Bonferroni correction for multiple comparisons, and unpaired Student's t-test if applicable. Statistical significance was accepted for P<0.05.



   Results
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Systemic characteristics
Total and LDL cholesterol levels were significantly elevated in the HC and HC+simvastatin groups (Table 1Go) compared with normal, but there were no differences between HC and HC+simvastatin. There were no significant differences among the groups in MAP or heart rate, or in plasma levels of sodium (P=0.4), potassium (P=0.7) or PRA (P=0.3) (Table 1Go). Serum creatinine levels tended to be slightly higher in both the HC and HC+simvastatin groups compared to normal, but this did not reach statistical significance (P=0.07) (Table 1Go). Plasma isoprostanes were significantly increased in the HC compared with the normal group, but were not different from normal in the HC+simvastatin group (Table 1Go).


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Table 1.  Systemic, urinary and EBCT-derived intra-renal haemodynamic characteristics of normal, high cholesterol (HC) and simvastatin-treated HC pigs (HC+S) after a 12-week diet

 

Renal morphology
Trichrome staining was significantly increased in HC compared to normal (Table 2Go, P=0.003), showing mild perivascular and focal interstitial fibrosis with associated tubular atrophy, but was significantly decreased in HC+simvastatin (Table 2Go, P=0.03 vs HC, Figure 1AGo).


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Table 2.  Morphological evaluation and immunostaining (% of renal area; mean±SEM) in normal, HC and HC+S kidneys

 


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Fig. 1.  Representative immunostaining of (A) tubuloglomerular trichrome, (B) eNOS and (C) nitrotyrosine in normal, HC and HC+simvastatin kidneys, respectively, showing significantly decreased glomerular and tubular fibrosis in the simvastatin-treated group, accompanied by increased availability of nitric oxide (x10).

 

Immunohistochemistry
Immunoreactivity of eNOS was present in renal arteriolar endothelial cells in all three groups (Figure 1BGo), but significantly attenuated in HC compared to normal (Table 2Go, P<0.001). However, it was largely restored in the HC+simvastatin group (Table 2Go, P<0.0003 vs HC), suggesting increased potential for NO production. Furthermore, these findings were accompanied in HC by enhanced immunoreactivity for nitrotyrosine (the marker for peroxynitrite formation in vivo), suggesting NO degradation via reaction with superoxide anion. However, this was normalized in HC+simvastatin, implying decreased production of superoxide and consequently increased bioavailability of NO by simvastatin (Figure 1CGo). The intensity of iNOS staining was significantly higher in HC compared with normal (Figure 2AGo, Table 2Go, P<0.01), indicating tubular proinflammatory changes. However, treatment with simvastatin largely restored iNOS expression in the HC+simvastatin group (Table 2Go, P=0.001 vs HC). In addition, expression of the proinflammatory transcription factor NF{kappa}B was increased in HC compared to normal (Figure 2BGo, Table 2Go, P<0.001), but was also mostly restored in HC+simvastatin (Table 2Go, P<0.03 vs HC). Moreover, the expression of the ox-LDL receptor LOX-1 was increased in HC and normalized in HC+simvastatin, suggesting decreased potential for uptake of ox-LDL (Figure 2CGo, Table 2Go, P=0.5 vs normal).



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Fig. 2.  Representative immunostaining of (A) iNOS, (B) NF{kappa}B and (C) LOX-1 in normal, HC and HC+simvastatin kidneys, respectively, and showing a significant decrease in tubular and glomerular inflammation in the simvastatin-treated group, accompanied by a decrease in ox-LDL uptake (x10).

 

Renal haemodynamics and function
Cortical perfusion was similar in the normal and HC groups (Figure 3Go). However, in the HC+simvastatin group, cortical and medullary perfusion and RBF were significantly elevated in comparison to both normal and HC (P<0.05, Table 1Go, Figure 3GoGo). GFR and renal volume were not significantly different among the groups (P=NS, Table 1Go).



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Fig. 3.  EBCT-derived basal cortical (solid bars) and medullary perfusion (dotted bars) in normal, HC, and HC pigs treated with simvastatin. *P<0.05 vs normal; {dagger}P<0.05 vs HC.

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The current study demonstrates that the HMG-CoA-reductase inhibitor simvastatin increases eNOS and decreases LOX-1 expression in the renal endothelial cells, decreases iNOS, nitrotyrosine, and NF{kappa}B immunoreactivity in tubular cells, and decreases tubulointerstitial and glomerular trichrome staining. Furthermore, these beneficial effects on the renal tissue are associated with augmentation of cortical and medullary perfusion in vivo. Hence, simvastatin decreases renal oxidative injury, inflammation and fibrosis, and enhances renal haemodynamics. Notably, these changes occurred in the absence of cholesterol lowering, and suggest direct beneficial effects of simvastatin on the kidney in HC.

Experimental evidence has suggested that lipid abnormalities play an important role in the progression of renal disease [1,2]. Statins are widely used for the treatment of HC, and clinical trials have consistently demonstrated their efficacy in reducing both LDL levels and the risk of major coronary events [7]. Some of their observed benefits exceeded the degree of lipid lowering [9,13], suggesting that statins have direct, lipid-lowering-independent cardiovascular effects [7,14]. Furthermore, previous studies have demonstrated that lipid-lowering doses of statins blunted renal insufficiency in a variety of experimental models of advanced renal disease [15], in association with a marked decrease in both blood pressure and plasma cholesterol [10, 1618]. However, in the absence of pre-existing renal disease, HC is initially associated with milder forms of renal injury. Nevertheless, thus far the direct effect of the statins on renal haemodynamics and function in HC has not been examined.

Our study demonstrated that simvastatin had salutary effects on the kidney in the HC swine model following 12 weeks of chronic administration, even in the absence of any significant lipid lowering. The dosing in this study was based on clinical practice in humans, in whom 80 mg/day is the maximal recommended dose [19], and on previous animal studies showing beneficial vascular effects at this dose, independent of lipid lowering [9,20,21]. Although the lipoprotein profile in swine is similar to that in humans [22], statins are less efficacious for decreasing lipid levels in this model [21], thus providing an opportunity to explore their lipid-lowering-independent mechanisms of action [9]. Indeed, our data are in line with a growing body of literature suggesting that the statins have direct protective effects independent of their lipid-lowering qualities [13,14], although their clinical relevance is still debated [6]. Our study demonstrates that in the kidney these effects include decreased renal oxidative injury, inflammation and fibrosis, with an associated increase in renal perfusion.

The underlying mechanisms leading to an increase in regional single-kidney renal perfusion may be multifactorial. The current study indicates that statins prevent the HC-induced decrease in eNOS in the renal vasculature, possibly via decreased uptake of ox-LDL, and decreased NO degradation by the superoxide anion. A subsequent increase in the bioavailability of NO due to both increased synthesis and decreased degradation may have contributed to the observed increase in regional renal perfusion compared to both HC and normal. Furthermore, the observed decreased immunoreactivity of nitrotyrosine in HC+simvastatin suggests decreased in vivo formation of peroxynitrite, which is a potent pro-oxidant and toxic substance per se. In addition, a decrease in plasma levels of isoprostanes, potent renal vasoconstrictors [12] and in vivo markers of increased oxidative stress, could also play a role in the increased renal perfusion. The concurrent increase in vasodilator capacity and decrease in vasoconstrictor activity might have led to the observed increase in cortical and medullary perfusion in simvastatin-treated HC animals. Although renal perfusion in HC pigs was similar to normal, HC is characterized by maladjusted response to challenge, and an increase in perfusion may hence decrease renal sensitivity to insults [5].

The statins also inhibit mesangial cell proliferation [23] and renal inflammation [24]. We observed increased expression of iNOS in HC, which in the kidney is involved in inflammation [5], and the increased tubular expression in HC possibly indicates early renal injury. Furthermore, iNOS can also be induced by NF{kappa}B [25], a transcription factor that regulates expression of numerous genes involved in inflammation and cell proliferation and is also increased in tubuloglomerular compartments in our HC group. Remarkably, our study demonstrates that simvastatin significantly attenuated both iNOS and NF{kappa}B expression in diet-induced HC, indicating a decrease in tubular inflammation. This was also accompanied by significant attenuation of glomerular and tubulointerstitial fibrosis in HC+simvastatin, suggesting an overall decrease in renal injury.

An additional mechanism by which HC induces renal injury is through cytotoxic effects of ox-LDL, since diet-induced HC is associated with a significantly increased propensity of LDL for oxidation [5], and LOX-1 subsequently facilitates the uptake and cytotoxic effects of ox-LDL. Recently, statins have been shown to decrease the expression of LOX-1 in vitro [26]. This study shows for the first time that diet-induced HC increases the expression of LOX-1 in renal arterioles, and simvastatin significantly attenuates the expression of this receptor in vivo, which might therefore represent a novel mechanism for the renal protective effects of simvastatin.

In addition to the effects that we observed, the pro-inflammation and vasoconstriction effects of HC may be mediated through alteration in the angiotensin II signalling pathway and increased expression of the AT1 receptor [27], and recent studies showed that statins down-regulated AT1 mRNA expression and modulated renal angiotensin II activity [18]. Blockade of the renin–angiotensin system may have also contributed to the increase in basal RBF observed in the treated group, but the mechanism of this effect will need to be investigated in animals supplemented with statins in the absence of HC. In fact, the capability of statins to reverse or arrest renal injury after HC has already been established needs to be investigated as well. Furthermore, it is possible that yet greater benefits can be achieved with a decrease in serum cholesterol, or longer treatment.

In summary, the current study demonstrates that simvastatin decreased inflammation and fibrosis in renal tissue, and augmented renal perfusion in the intact kidney of pigs with diet-induced HC, associated with decreased oxidative stress. These changes were observed in the absence of cholesterol lowering, and may reflect a novel renoprotective effect of HMG-CoA-reductase inhibitor in the setting of HC and atherosclerosis.



   Acknowledgments
 
This study was supported in part by grant HL-63282 from the NIH, by the Mayo Foundation, by an unrestricted Medical School grant from Merck, and the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labor and Welfare of Japan; the Organization for Pharmaceutical Safety and Research; Takeda Science Foundation; and Ono Medical Research Foundation (T.S.).

Conflict of interest statement. None declared.



   Notes
 
Correspondence and offprint requests to: Lilach O. Lerman, MD, PhD, Division of Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Email: Lerman.lilach{at}mayo.edu Back



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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 29. 8.02
Accepted in revised form: 23.10.02