J Mol Hist (2011) 42:323–331 DOI 10.1007/s10735-011-9336-4
ORIGINAL PAPER
Rosuvastatin beneficially alters the glomerular structure of kidneys from spontaneously hypertensive rats (SHRs) E´rica Peres de Barros • Ange´lica Beatriz Garcia-Pinto • Priscilla Yo´rio Machado • Ma´rio Jose´ dos Santos Pereira Jorge Jose´ de Carvalho
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Received: 19 April 2011 / Accepted: 30 May 2011 / Published online: 14 June 2011 Ó Springer Science+Business Media B.V. 2011
Abstract The incidence of chronic renal diseases is increasing worldwide, and there is a great need to identify therapies capable of arresting or reducing disease progression. The current treatment of chronic nephropathies is limited to angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, but increasing clinical and experimental evidence suggests that statins could play a therapeutic role. Ultrastructural studies have shown the presence of gap junctions within all the cells of the glomerulus and podocytes have been found to contain primarily connexin-43. The present study aims to observe the beneficial effects of rosuvastatin on structural and ultrastructural renal morphology and on glomerular connexin43 expression in normotensive rats and spontaneously hypertensive rats (SHR). Rats were randomly allocated into four groups: WKY-C: normotensive animals no receiving
E´rica Peres de Barros and Ange´lica Beatriz Garcia-Pinto contributed equally for this work. E´. P. de Barros A. B. Garcia-Pinto (&) P. Y. Machado J. J. de Carvalho Laborato´rio de Ultraestrutura e Biologia Tecidual, Departamento de Histologia e Embriologia, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro, 87, Rio de Janeiro, RJ 20551-030, Brazil e-mail:
[email protected] E´. P. de Barros A. B. Garcia-Pinto Programa de Po´s-Graduac¸a˜o em Fisiopatologia Clı´nica e Experimental, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil M. J. dos Santos Pereira Laborato´rio de Fisiologia da Nutric¸a˜o e Desenvolvimento, Departamento de Fisiologia, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil
rosuvastatin; WKY-ROS: normotensive animals receiving rosuvastatin; SHR-C: hypertensive animals no receiving rosuvastatin; SHR-ROS: hypertensive animals receiving rosuvastatin. Our results show no differences in blood urea, creatinine, uric acid and creatine phosphokinase levels between the groups, however, there was an decreasing of 24-h protein excretion in SHR-ROS. Capsular area in SHRROS was decreased, however, there was no alteration in urinary space. By transmission electron microscopy the slit diaphragm and podocyte foot processes were more preserved in SHR-ROS. By scanning electron microscopy the podocyte foot processes were more preserved in SHR-ROS. Increased connexin-43 immunofluorescence was observed in glomeruli of WKY-ROS and SHR-ROS. In conclusion, we hypothesize that renal pleiotropic effect of rosuvastatin can be a therapeutic tool for improving kidney ultrastructure and, consequently, renal function in hypertensive individuals. Keywords Rosuvastatin Ultrastructure Connexin-43 Kidney Hypertension SHR
Introduction Cardiovascular disease is a burden on worldwide healthcare, accounting for about 50% of deaths in developed countries and 25% of deaths in developing countries. Cardiovascular disease includes a variety of disorders, including coronary heart disease (myocardial infarction, angina and coronary insufficiency), stroke, congestive heart failure, peripheral vascular disease and hypertension (Pfefferkorn 2011). Hypertension leads to targeted organ damage of the heart, blood vessels, brain, eyes and kidneys with eventual
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development of cardiovascular and renal diseases and is also a well-known risk factor for the rapid progression of kidney failure (Barri 2008; Krzesinski and Cohen 2007). The incidence of chronic renal diseases is increasing worldwide, and there is a great need to identify therapies capable of arresting or reducing disease progression. The current treatment of chronic nephropathies is limited to angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, but increasing clinical and experimental evidence suggests that statins (3-hydroxy-3-methylglutaryl-CoA reductase inhibitors) could play a therapeutic role (Bae et al. 2009; Gianella et al. 2007). Statins are some of the most widely prescribed medications in the United States for treating dyslipidemia (Pfefferkorn 2011). Their benefits are derived both from reducing atherogenic lipoprotein levels (LDL-C) and from increasing antiatherogenic lipoproteins (HDL-C) (Nicholls et al. 2007). Besides modulating lipids, recent clinical and experimental studies have shown that statins have pleiotropic effects, such as anti-inflammatory, anti-proliferative, and anti-thrombotic effects, attenuation of NADPH oxidase-mediated superoxide generation and improving endothelial vasomotor function, all of which may affect cardiovascular outcomes in high-risk patients (Suh et al. 2010). In addition to these effects on the cardiovascular system (Neto-Ferreira et al. 2011), statins exert effects in muscles (Hedenmalm et al. 2010), bones (Chen et al. 2010) and kidneys (Renke et al. 2010). The administration of various statins has been reported to exhibit beneficial effects in a number of experimental models of chronic kidney diseases suggesting that lipids may be important therapeutic targets to halt or attenuate renal injury (Strippoli et al. 2008). Although studies have evaluated the effects of statins on the progression of chronic kidney disease, the results are controversial (Gianella et al. 2007; Strippoli et al. 2008; Verhulst et al. 2008). Although it is difficult to distinguish the effects that are dependent or independent of their cholesterol-lowering effects in clinical trials, emerging evidence suggests that the renoprotection provided by statins is due to their pleiotropic properties (Gianella et al. 2007; CormackAboud et al. 2009; Bae et al. 2009). Vertebrate cells communicate in part by sharing ions, second messengers, small metabolites and other signaling molecules through gap junctions. This type of intercellular communication permits coordinated cellular activity, including secretion, by allowing cells to review the functional state of their neighbors, a critical feature for the homeostasis of multicellular systems. Ultrastructural studies in both humans and rats have shown the presence of gap junctions within all cells of the glomerulus, and the podocytes primarily contain connexin-43 (Cx-43) (Hanner et al. 2010).
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In the present study, we investigated the beneficial effects of rosuvastatin on structural and ultrastructural renal morphology and on glomerular Cx-43 expression in normotensive rats and spontaneously hypertensive rats (SHRs) without cholesterol disturbance.
Materials and methods Animals and treatments This study was performed in accordance with the guidelines of the ‘‘Care and Use of Laboratory Animals’’ (US National Institutes of Health 85-23, revised 1996). The handling and experimentation protocols were approved by the Universidade do Estado do Rio de Janeiro Ethics Committee for the Use and Care of Experimental Animals. Wistar-Kyoto rats (WKY) and SHRs (lzm strain) were kept under standard conditions (12 h light/dark cycle, 21 ± 2°C, humidity 60 ± 10%) and received water and standard chow ad libitum (Nuvilab, Parana, Brazil). Twomonth-old rats were purchased from the Center of Experimental Models for Medicine and Biology, Universidade Federal de Sa˜o Paulo (www.unifesp.br/centros/cedeme) and the experiment began when rats were 5-months-old. Rats were randomly allocated into four groups (n = 8 each): WKY-C (control): normotensive animals not receiving rosuvastatin; WKY-ROS: normotensive animals receiving 20 mg/kg/day rosuvastatin (Crestor, AstraZeneca, Brazil) by orogastric gavage; SHR-C (hypertensive control): hypertensive animals not receiving rosuvastatin; and SHR-ROS: hypertensive animals receiving rosuvastatin, as described for the WKY-ROS group. The experiment lasted 5 weeks. Blood pressure (BP) and body mass (BM) were measured weekly. The BP was measured in conscious rats by the non-invasive tail-cuff plethysmography method (Leica LE 5100, Panlab, Spain). Urinary excretion, biochemical analysis of blood and euthanasia At week 6, the 24 h urine was collected in a metabolic cage, and urinary protein excretion was determined with a Urea UV Kit (Kovalent, Sa˜o Gonc¸alo, Brazil). The rats were deeply anesthetized with sodium pentobarbital (i.p., 150 mg/kg), and the animals were euthanized with excess anesthetic. Heparinized whole blood was withdrawn by cardiac puncture from rats and immediately used to determine urea, creatinine, uric acid and creatine phosphokinase (CPK) as biomarkers of renal function. After these procedures, the abdomen was opened, and the kidneys were removed.
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Morphometry Five sagittal sections per rat were obtained, and the tuft area of each individual glomerular profile was delineated and measured in lm2 with an image analyzer using Image Pro Plus 4.5. Superficial glomeruli were considered as those located up to 250 lm from the capsule (Ossani et al. 2009). A total of 4,000 glomeruli were measured.
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variances, and then differences between groups were analyzed by one-way ANOVA analysis of variance and posthoc Tukey’s test. Two-way analysis of variance was also performed, considering the effect of genotype (WKY or SHR). A P-value B0.05 was considered statistically significant (Graph-Pad Prism version 5.03, San Diego, USA).
Results Transmission electron microscopy Blood pressure and body mass Fragments of the kidneys were immediately fixed in 2.5% glutaraldehyde (Sigma–Aldrich Laborchemikalien GmbH, Seelze, Germany) in a 0.1 M cacodylate buffer (pH 7.2) and 0.25% tannic acid (Merck KGaA, Darmstadt, Germany), post-fixed in 1% osmium tetroxide (SigmaAldrich, Saint Louis, USA), and then embedded in Epon (Embed-812, EMS, Hatfield, PA, USA). Ultrathin sections (60–70 nm) were obtained from selected areas with an ultramicrotome (Leica ULTRA-CUT; Leica Aktiengesellschaft, Wien, Austria), stained with uranyl acetate and lead citrate, and then examined with a Zeiss EM 906 transmission electron microscope (TEM) (Carl Zeiss EM 906, Oberko¨chen, Germany) at 80 kV. Scanning electron microscopy Other fragments of the kidneys were fixed in 2.5% glutaraldehyde (Sigma–Aldrich Laborchemikalien GmbH, Seelze, Germany) in a 0.1 M cacodylate buffer (pH 7.2) and post-fixed for 30 min in 1% osmium tetroxide and dehydrated acetone. Samples were then critical point-dried with CO2, coated with gold, and examined with a scanning electron microscope (SEM; Carl Zeiss LEO 1450 VP, Oberko¨chen, Germany) at 15 kV.
The blood pressure (BP) of the WKY-C and WKY-ROS groups did not differ significantly (P [ 0.05) throughout the experiment (Fig. 1). The SHR-C and SHR-ROS groups had increased BP levels (from 163 ± 15 to 202 ± 9 and from 155 ± 12 to 207 ± 7, respectively) (P \ 0.05) after 5 weeks of the experiment and began significantly changing at the 3rd week. The BM of the WKY-C and WKY-ROS groups and of the SHR-C and SHR-ROS groups did not differ significantly (P [ 0.05) throughout the experiment (Fig. 2). However, we observed differences between WKY and SHR before treatment and after 5 weeks of treatment with rosuvastatin (P \ 0.001). In general, the animals showed a gradual increase in BM. Urinary excretion and biochemical analysis of blood No differences in blood urea, creatinine, uric acid and CPK levels were observed among the groups (P [ 0.05) (Table 1). The 24-h protein excretion was increased in SHR-C in comparison with that in WKY-C and WKY-ROS (P \ 0.01). Although SHR-ROS exhibited decreased
Immunofluorescence Kidney sections were fixed in a fixative solution (freshly prepared 1.27 mol/l formaldehyde in a 0.1 M phosphate buffer; pH 7.2), washed and incubated overnight with mouse anti-connexin-43 (anti-Cx-43) (1:20, MAB3067, Chemicon) monoclonal primary antibodies, followed by incubation with Alexa FluorÒ-488 donkey anti-mouse (A21202, Invitrogen) secondary antibody and staining with Evans blue dye 1:5000 for 15 min. The sections were mounted and observed under a Zeiss LSM 510 META laser scanning confocal microscope. Data analysis Data are shown as the mean and standard error of the mean. Data were tested for normality and homogeneity of
Fig. 1 Blood pressure before and during 5 weeks of treatment. C control, SHR spontaneously hypertensive rats, ROS rosuvastatin, WKY Wistar-Kyoto rats. One way ANOVA and post hoc Tukey’s test; P \ 0.001, when: a versus WKY-C, b versus WKY-ROS
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Fig. 2 Body Mass before and during 5 weeks of treatment. C control SHR spontaneously hypertensive rats, ROS rosuvastatin, WKY WistarKyoto rats. One way ANOVA and post hoc Tukey’s test; P \ 0.001, when: a versus WKY-C, b versus WKY-ROS
protein excretion, this result was not significant when compared to any of the other groups (P [ 0.05) (Fig. 3).
Fig. 3 Urinary protein excretion. The 24-h protein excretion was increased in SHR-C (*) in comparison with that in WKY-C and WKY-ROS. Although SHR-ROS exhibited decreased protein excretion, this result was not significant when compared to any of the other groups. C control, SHR spontaneously hypertensive rats, ROS rosuvastatin, WKY Wistar-Kyoto rats. (*) for P \ 0.01 when compared to control group
Morphometry
Immunofluorescence
No differences (P [ 0.05) were observed in the urinary space among the studied groups (Fig. 4a). The capsular area increased in SHR-C in comparison with WKY-C and WKY-ROS (P \ 0.05). Despite SHRROS exhibiting a decreased capsular area, this result was not significant when compared to any of the other groups (P [ 0.05) (Fig. 4b).
Increased Cx-43 immunofluorescence was observed in visceral epithelial cells from WKY-ROS glomeruli (Fig. 7b) and in parietal and visceral epithelial cells of SHR-ROS glomeruli (Fig. 7d) when compared to the WKY-C (Fig. 7a) and SHR-C (Fig. 7c) groups.
Discussion Transmission electron microscopy In the glomerular filtration barrier, the slit diaphragm and podocyte foot processes (pedicel) were more preserved in WKY-C (Fig. 5a), WKY-ROS (Fig. 5b) and SHR-ROS (Fig. 5d). In SHR-C (Fig. 5c), podocytes exhibited vacuoles and effacement of the pedicels. Scanning electron microscopy Pedicels were preserved in WKY-C (Fig. 6a), WKY-ROS (Fig. 6b) and SHR-ROS (Fig. 6d). However, the pedicels were thinner, shorter and disorganized in SHR-C (Fig. 6c).
The aim of the present study was to provide new information about the development of kidney damage in hypertensive animals and to determine if the pleiotropic effects of rosuvastatin are beneficial to these animals. Loch et al. (2006) investigated the pleiotropic effects of statins as a potential mechanism for the treatment of heart damage in hypertension. They described that rosuvastatin therapy attenuated the development of cardiovascular hypertrophy, inflammation, fibrosis, and ventricular action potential prolongation, but did not modify hypertension or vascular dysfunction and concluded that the pleiotropic effects of rosuvastatin include attenuation of aspects of
Table 1 Effects of the rosuvastatin on the blood urea, creatinine, uric acid and creatine phosphokinase (CPK) from SHR Groups (n = 8)
Urea (mg/dl)
Creatinine (mg/dl)
Uric acid (mg/dl)
CPK (U/I)
WKY-C
45.1 ± 1.17
0.5 ± 0.01
1.3 ± 0.06
876.7 ± 131.62
WKY-ROS
44.5 ± 0.89
0.5 ± 0.01
1.1 ± 0.02
662.7 ± 73.27
SHR-C
48.3 ± 1.10
0.5 ± 0.01
1.3 ± 0.09
866.8 ± 65.72
SHR-ROS
50.8 ± 1.39
0.4 ± 0.01
1.3 ± 0.11
603.0 ± 48.70
No statistically differences were found between the studied groups (P [ 0.05) C control, SHR spontaneously hypertensive rats, ROS rosuvastatin, WKY Wistar-Kyoto rats
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Fig. 4 Morphometry of the glomerular structures. No differences were observed in the urinary space among the studied groups (a). The capsular area increased in SHR-C in comparison with WKY-C and WKY-ROS (b). Despite SHR-ROS exhibiting a decreased capsular
area, this result was not significant when compared to any of the other groups (b). C control, SHR spontaneously hypertensive rats, ROS rosuvastatin, WKY Wistar-Kyoto rats
Fig. 5 Transmission electron micrograph of a glomerulus. In the glomerular filtration barrier, the slit diaphragm (arrow) and podocyte foot processes (pedicel) (a) were more preserved in WKY-C (a), WKY-ROS (b) and SHR-ROS (d). In SHR-C (c), podocytes exhibited
vacuoles and effacement of the pedicels. a WKY-C (215609), b WKY-ROS (215609), c SHR-C (167009), d SHR-ROS (215609). Abbreviation: C control, SHR spontaneously hypertensive rats, ROS rosuvastatin, WKY Wistar-Kyoto rats
cardiovascular remodeling in the DOCA-salt model of hypertension in rats without altering systolic blood pressure. These findings are in agreement with our results
which show there has been no change in blood pressure of normotensive or hypertensive rats treated or not with rosuvatatin.
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Fig. 6 Scanning electron micrograph. In the glomerular filtration barrier, the podocyte foot processes (pedicel) (arrow) were more preserved in WKY-C (a), WKY-ROS (b) and SHR-ROS (d). However, the pedicels were thinner, shorter and disorganized in
SHR-C (c). a WKY-C (240009), b WKY-ROS (240009), c SHR-C (160009), d SHR-ROS (240009). C control, SHR spontaneously hypertensive rats, ROS rosuvastatin, WKY Wistar-Kyoto rats
In damaged kidneys, the myogenic reflex is blunted, renal autoregulation becomes impaired, and the ability to prevent the transmission of systemic BP changes to the glomerular circulation is partially or completely lost. Consequently, intraglomerular pressure begins to change in direct proportion to changes in systemic arterial pressure (Palmer and Fenves 2010). Remodeling in the resistance vessels exposed to increased pressures causes benign nephrosclerosis over time. However, when the BP exceeds the threshold for autoregulation and the risk of vascular injury is increased, acute malignant nephrosclerosis ensues and the autoregulatory ability of the pre-glomerular vasculature to protect the glomerular capillaries is breached (Bidani et al. 2009). Renal vascular resistance is increased in SHRs, possibly as a result of smaller afferent arterioles and probably also as a consequence of physiological autoregulation after the onset of hypertension (Ren et al. 2010; Ofstad and Iversen 2005). Using TEM, we observed that the slit diaphragm and the pedicels were more preserved in the glomerular filtration barrier of WKY-C, WKY-ROS and SHR-ROS. In contrast, the SHR-C exhibited vacuoles in podocytes and effacement of the pedicels. Increasing intraglomerular pressure can possibly account for how hypertension affected the glomerular ultrastructure of SHR-C kidneys. To better understand the changes in the pedicels, we used SEM. We observed that the pedicels were preserved in WKY-C, WKY-ROS and SHR-ROS. However, the
pedicels were thinner, shorter and disorganized in SHR-C. These observations are in agreement with rosuvastatin significantly reducing podocyte-induced apoptosis, suggesting that statins may have a generalized prosurvival effect on podocytes by acting in a p21-dependent fashion. Cytoplasmic p21 has been shown to bind to and prevent activation of procaspase-3, thus interfering with Fas-mediated apoptosis (Cormack-Aboud et al. 2009). Changes in the glomerular filtration barrier can cause proteinuria (Tryggvason et al. 2006). This barrier has three layers: the fenestrated endothelium, the glomerular basement membrane, and the podocytes. The podocyte slit diaphragm has an important and direct role in glomerular filtration. Some of its protein components are involved in proteinuria (Putaala et al. 2001; Donoviel et al. 2001; Ciani et al. 2003; Roselli et al. 2004). In our experiments, we observed that some components of the glomerular filtration barrier, such as the slit diaphragm and the pedicels, are altered in SHR-C. Proteinuria, a useful marker of kidney damage associated with hypertension, is itself a risk factor for the progression of renal disease (Atkins et al. 2005; Flack et al. 2010). As expected, we observed increased 24-h protein excretion in SHR-C compared to WKY-C and WKY-ROS. Despite SHR-ROS exhibiting decreased protein excretion, this result was not significant when compared with any of the other groups. No differences were significant in blood urea, creatinine, uric acid and CPK levels among the groups, although structural changes may
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Fig. 7 Connexin-43 immunofluorescence of glomeruli. Increased Cx-43 immunofluorescence (arrow) was observed in visceral epithelial cells from WKY-ROS glomeruli (b) and in parietal and visceral epithelial cells of SHR-ROS glomeruli (d) when compared to the
WKY-C (a) and SHR-C (c) groups. a WKY-C (609), b WKY-ROS (609), c SHR-C (609), d SHR-ROS (609). C control, SHR spontaneously hypertensive rats, ROS rosuvastatin, WKY WistarKyoto rats
have begun before the biochemical disturbances. The beneficial effects of rosuvastatin observed in this study were at least partly attributable to significant antiproteinuric action because of the preservation of podocyte integrity (as shown by our ultrastructural findings). The total glomerular filtration rate depends upon the structural integrity and the area of the glomeruli. Measuring glomerular area or volume is widely employed in human and experimental biology (Ossani et al. 2009). In our experiments, we observed no differences in the area of urinary space among the studied groups. The SHR-C exhibited increased capsular area compared to WKY-C and WKY-ROS. Despite SHR-ROS showing a decreased capsular area, this result was not significant when compared to any of the other groups. The intercellular gap junction results from the association of 2 half channels, named connexons. Each connexon is an assembly of 6 membrane proteins, named connexins.
Ultrastructural studies in both humans and rats have shown the presence of gap junctions within all cells of the glomerulus, and podocytes have been found to primarily contain Cx-43 (Hanner et al. 2010). Dlugosova et al. (2009) noticed a significant improvement in aortic wall from SHR as a result of increased expression of Cx-43 by omega-3 fatty acid diet. Increased Cx-43 immunofluorescence was observed in the visceral epithelial cells of WKY-ROS glomeruli and in parietal and visceral epithelial cells of the SHR-ROS glomeruli compared to WKY-C and SHR-C groups. Increased immunofluorescence of Cx-43 in WKYROS and SHR-ROS groups indicated that rosuvastatin affected Cx-43 expression in hypertensive and normotensive animals during the study period. Yaoita et al. (2002) described an increase of Cx-43 expression in the earliest response in podocyte injury and concluded that Cx-43 is expressed in podocyte injury in the integrated epithelium on a glomerulus rather than in individual cells. We
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hypothesize that the increased expression of Cx-43 in renal glomeruli of SHR is a beneficial response in result the action of rosuvastatin. Although the role of Cx-43 in the kidneys is not well defined, we believe that this increased expression of connexin result in increased quality of signaling and interaction between the glomerular cells. Our results show the effects of hypertension on kidney ultrastructure and the benefits of rosuvastatin for hypertensive individuals. In conclusion, we suggest that the renal pleiotropic effect of rosuvastatin can be used as a therapeutic tool for improving kidney ultrastructure and consequently renal function in hypertensive individuals. Acknowledgments This study was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Fundac¸a˜o Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de nı´vel Superior (Capes) and Universidade do Estado do Rio de Janeiro (UERJ).
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