Journal of the American Society of Nephrology
2007 JASN IMPACT FACTOR 7.111 HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP 
    advanced
CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Attia, D. M.
Right arrow Articles by Joles, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Attia, D. M.
Right arrow Articles by Joles, J. A.
J Am Soc Nephrol 15:949-957, 2004
© 2004 American Society of Nephrology

BASIC SCIENCE

Hypercholesterolemia in Rats Induces Podocyte Stress and Decreases Renal Cortical Nitric Oxide Synthesis via an Angiotensin II Type 1 Receptor–Sensitive Mechanism

Diana M. Attia*, Olivier Feron{dagger}, Roel Goldschmeding{ddagger}, Louk H. Radermakers{ddagger}, Nosratola D. Vaziri§, Peter Boer*, Jean-Luc Balligand{dagger}, Hein A. Koomans* and Jaap A. Joles*

*Department of Nephrology and Hypertension, University Medical Center, Utrecht, the Netherlands; {dagger}Department of Internal Medicine, Louvain University, Brussels, Belgium; {ddagger}Department of Pathology, University Medical Center, Utrecht, the Netherlands; and §Division of Nephrology, Departments of Medicine, Physiology, and Biophysics, University of California Irvine, Irvine, California

Correspondence to Dr. Jaap A. Joles, Department of Nephrology and Hypertension (Room F03.226), University Medical Center, Heidelberglaan 100, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. Phone: 31-30-2507329; Fax: 31-30-2543492; E-mail: J.A.Joles{at}med.uu.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Podocyte stress precedes proteinuria in hypercholesterolemic rats. Molsidomine, a nitric oxide (NO) donor, prevented podocyte stress and proteinuria in long-term hypercholesterolemia, suggesting that podocyte stress was due to NO deficiency. Podocytes express the angiotensin II type 1 receptor, which influences their function. Because NO counteracts angiotensin II, it was hypothesized that in a setting of impaired renal NO availability, angiotensin II receptor inhibition could prevent podocyte stress. For determining the effect of NO deficiency on podocyte stress, one group of female rats were fed 2% cholesterol and another group the arginine analogue N-{omega}-nitro-L-arginine (L-NNA; 40 mg/kg food) for 2 wk. Another group of rats that were fed 2% cholesterol also received the NO donor molsidomine (120 mg/L water) for 2 wk before and during cholesterol feeding. For determining the influence of angiotensin II in the setting of decreased renal NO availability, rats that were treated with cholesterol or L-NNA received the angiotensin II type 1 antagonist losartan (200 mg/L water) for 2 wk before and during cholesterol or L-NNA administration. Desmin staining and electron microscopy were used to monitor podocyte activation. Glomerular caveolin was quantified by immunohistochemistry. Renal cortical NO synthesis, NO synthase isoforms, and caveolin-1 protein mass were also measured. Both short-term cholesterol and L-NNA induced podocyte stress as evidenced by enhanced desmin staining and electron-dense fused foot processes. Podocyte stress was prevented by molsidomine in short-term hypercholesterolemia. Furthermore, losartan prevented podocyte stress in rats that were treated with cholesterol or with L-NNA. Finally, hypercholesterolemia decreased renal cortical NO synthase activity and increased caveolin-1 protein mass and glomerular caveolin staining, and these changes were also prevented by losartan. It is suggested that podocyte stress in these models of early injury results from angiotensin II, unopposed by the action of endogenous NO. This underscores the strategic role of angiotensin II blockers in early kidney disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hypercholesterolemia has been found to decrease nitric oxide (NO) availability in the renal circulation (1) and renal artery (2). In a previous study, we showed that hypercholesterolemia can decrease renal NO synthase (NOS) activity and cause podocyte stress before proteinuria (3). We suggested that podocyte stress could be due to NO deficiency, because the NO donor molsidomine (4) prevented the long-term effects of cholesterol loading. Therefore, our first question was whether molsidomine could also prevent early hypercholesterolemia-induced podocyte stress.

Angiotensin-converting enzyme (ACE) inhibitors and angiotensin II type 1 (AT-1) receptor blockers prevent hypercholesterolemia-induced vascular injury in various species (5–10). Similarly, angiotensin II receptor blockade can prevent long-term effects of hypercholesterolemia on proteinuria and renal injury. For instance, treatment of hypercholesterolemic rats with an ACE inhibitor (11) or hypercholesterolemic Imai rats with an AT-1 receptor antagonist (12) prevented the development of glomerulosclerosis. The protective effects of angiotensin II receptor blockade are thought to occur through inhibition of proteinuria and by diminishing interstitial inflammation, which are late effects. However, podocyte changes are early events. Podocytes contain AT-1 receptors, which upon stimulation increase intracellular calcium activity and, possibly, podocyte contractility (13). Furthermore, stimulation of cultured podocytes with angiotensin II increased cytoskeletal density (14). Angiotensin II infusion in vivo causes podocyte injury (15), and this can be prevented by AT-1 receptor blockade (16). In the kidney, NO counteracts angiotensin II (17,18), as is the case in the systemic circulation (19). Thus, when hypercholesterolemia or NOS inhibition reduces renal NO availability, angiotensin II may become injurious to podocytes. Therefore, the second question was whether AT-1 receptor blockade could prevent the early stress of NO deficiency, as a result of either cholesterol or NOS inhibition, in podocytes.

Finally, it has been shown that cholesterol increases caveolin-1 expression in endothelial cells, which binds to endothelial NOS (eNOS), thus inhibiting its activity (20). This notion is particularly interesting in view of the interaction of angiotensin II and hypercholesterolemia. Because angiotensin II is also known to increase caveolin-1 biosynthesis and turnover in caveolae-like invaginations in cultured vascular smooth muscle cells (21) and to decrease NOS expression and activity in cultured endothelial cells (22), it might be that prevention of cholesterol effects by angiotensin II receptor blockade focuses on caveolin-1 and its effect on NOS activity. Therefore, we investigated whether in the kidney angiotensin II and hypercholesterolemia interact at the level of caveolin-1 and NOS activity.

For excluding effects secondary to proteinuria, rats were exposed to NOS inhibition or hypercholesterolemia for only 2 wk. For examining the effect of NO deficiency on podocyte stress, rats received either a low dose of N-{omega}-nitro-L-arginine (L-NNA) or 2% dietary cholesterol. For examining whether early podocyte stress in cholesterol-fed rats could be prevented by exogenous NO administration, rats were fed 2% cholesterol and received the NO donor molsidomine. The deleterious effects of NO deficiency, in the absence of angiotensin II, were studied in rats that received an AT-1 receptor antagonist (losartan) for 2 wk before and during either L-NNA or cholesterol administration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female Sprague Dawley rats (150 to 175 g; Harlan-Olac, Blackthorn, UK) were exposed to a 12-h light/dark cycle, an ambient temperature of 22°C, and a humidity of 60%. Sentinel animals, which were monitored regularly for infection by nematodes and pathogenic bacteria, as well as for antibodies for a large number of rodent viral pathogens (International Council for Laboratory Animal Science, Nijmegen, The Netherlands), consistently tested negative for infection throughout the experiment. The Utrecht University Board for studies in experimental animals approved the studies.

Experimental Protocol 1: Hypercholesterolemia and Podocyte Stress
Rats were treated for 4 wk. Six groups of rats were studied, each group consisting of six to eight animals. The control group received regular diet and tap water. Water and food intake were ~80 ml/kg body wt per d and 70 g/kg body wt per d, respectively. The second group received molsidomine dissolved in drinking water (120 mg/L, resulting in a dose of ~10 mg/kg per d) for 4 wk. The third group received losartan dissolved in drinking water (200 mg/L, resulting in a dose of ~16 mg/kg per d). The fourth group was fed 2% cholesterol + 0.5% cholate for the last 2 wk, resulting in an intake of 1.4 g cholesterol/kg body wt per d. The fifth group received molsidomine for 4 wk and 2% cholesterol + 0.5% cholate for the last 2 wk. The sixth group received losartan for 4 wk and was fed 2% cholesterol + 0.5% cholate for the last 2 wk. Cholesterol and cholate were mixed through chow (RMH-TM; Hope Farms, Woerden, the Netherlands).

Experimental Protocol 2: NOS Inhibition and Podocyte Stress
Two additional groups were studied for 4 wk. The seventh group received L-NNA in food (40 mg/kg, resulting in a dose of ~3 mg/kg per d)) for the last 2 wk. The eighth group received losartan for 4 wk and L-NNA for the last 2 wk.

At the end of both protocols, blood was collected under anesthesia (see below), and the kidneys were removed and cut transversely into three slices. The middle slice was immersion-fixed in PBS formaldehyde (4%; pH 7.35) and embedded in paraffin for morphology and for desmin and caveolin staining. The poles were frozen in liquid nitrogen and stored at -80°C until being processed for renal cortical NOS activity, renal cortical NOS and caveolin-1 protein abundance, and glomerular lipid deposits. For transmission electron microscopy, kidneys of two cholesterol-fed and two control rats were perfused with appropriate fixative (see below) for 2 min at 160 mmHg.

Plasma Lipids, BP, Body Weight, and Renal Function
At the end of the experiment (week 4), the animals were anesthetized with 60 mg/kg sodium pentobarbital intraperitoneally to collect blood from the aorta for determination of plasma creatinine and lipids. Plasma cholesterol and triglycerides were determined enzymatically (Boehringer, Mannheim, Germany). Systolic BP (SBP) was measured in awake rats, starting 1 wk before the start of treatment (week 0) by the tail-cuff method (IITC, San Diego, CA). Urine was collected at weeks 0 and 4 for determination of urinary protein excretion. The rats were weighed and placed in metabolic cages for 24 h, with free access to food and water. Urinary protein levels were determined by the Bradford method. Plasma creatinine levels were determined colorimetrically (Sigma Diagnostics, St. Louis, MO).

Histochemistry
Glomerular lipid deposits were determined with Sudan II/IV on frozen kidney sections. The staining was scored semiquantitatively with x400 magnification by evaluating the percentage of the glomerulus showing positive staining, 0 = 0 to 5% stained; 1 = 5 to 25%; 2 = 25 to 50%; 3 = 50 to 75%; 4 = >75%. Twenty glomeruli were scored. A total score was determined by adding (0 x score 0) + (1 x score 1) + (2 x score 2) + (3 x score 3) + (4 x score 4).

Immunohistochemistry
Immunohistochemistry was carried out on 3-µm paraffin sections of formaldehyde-fixed kidney. Tissue sections were deparaffinized and dehydrated. Glomerular desmin staining was used to determine podocyte activation (23). Paraffin sections of formaldehyde-fixed kidney were stained with mu072-uc (Biogenex). The outer cell layer of the glomerular tuft was evaluated. The staining was scored semiquantitatively with x400 magnification by evaluating the percentage of the glomerular edge showing positive staining, 0 = 0 to 5% stained; 1 = 5 to 25%; 2 = 25 to 50%; 3 = 50 to 75%; 4 = >75%. Fifty glomeruli were scored. A total score was determined as described for glomerular lipid deposits. Glomerular caveolin was evaluated morphometrically. Endogenous peroxidase reactions were blocked with citric acid (40 mmol/L) plus Na2HPO4 (120 mmol/L; pH 5.8) for 15 min. Aspecific reactions were blocked with 10% normal goat serum in PBS for 15 min. Paraffin sections were incubated overnight with the primary rabbit anti-rat polyclonal caveolin antibody (BD Transduction Laboratories, San Jose, CA; 1:150 in 10% normal goat serum/PBS) at 4°C. The signal was enhanced by incubating for 30 min at room temperature with goat anti-rabbit Powervision (polymerized horseradish peroxidase–goat anti-rabbit; Immunologic, Duiven, The Netherlands). After rinsing for 5 min with acetate buffer (100 mmol/L; pH 4.8), color development was done with 3-amino-9-ethylcarbazole substrate (Sigma). After counterstaining with hematoxylin, sections were covered with paragon. The stained glomerular area was quantified morphometrically with Analysis software in 20 glomeruli/kidney at x400 magnification and expressed as a percentage of total glomerular area.

Transmission Electron Microscopy
For transmission electron microscopy, kidneys of two cholesterol-fed and two control rats were perfused with 2% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.4) containing 5 mM CaCl2 and 10 mM MgCl2. Small blocks of kidney cortex were cut and postfixed in 1% OsO4 in the same buffer for 2 h. After dehydration in a graded series of ethanol, the samples were embedded in Epon. Semithin 1-µm sections were cut with glass knives on a Reichart Ultracut ultramicrotome and stained with methylene blue and pararosanilin (24). From each kidney, three glomeruli, i.e., six per rat, were selected. Ultrathin sections were cut with a diamond knife on a Reichart Ultracut, stained with 6% uranyl acetate at 63°C for 45 min followed by lead citrate for 10 min, and evaluated in a Phillips JEOL JEM1010 electron microscope.

Renal Cortex NOS Activity
NOS activity was measured by determining the formation of L-3H-citrulline from L-3H-arginine. Using an Ultraturrax, an aliquot of ~300 mg kidney cortex tissue was homogenized in an ice-cooled homogenization buffer (pH 7.4) consisting of 50 mmol/L Tris buffer, 320 mmol/L sucrose, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 2 mg/L aprotinin, and 100 mg/L PMSF. An aliquot of 50 µl of homogenate was incubated in a final volume of 100 µl at 37°C for 30 min with 1 mmol/L L-citrulline, 0.3 mmol/L tetrahydrobiopterin, 300 U/ml calmodulin, 0.5 mmol/L NADPH, 1 mmol/L CaCl2, 0.01 mmol/L L-arginine, and 3.7 kBeq L-[2,3,4,5]-arginine (Amersham Pharmacia Biotech, Buckinghamshire, UK) in 50 mmol/L KH2PO4 phosphate buffer (pH 7.2). In an additional tube, the NADPH was substituted by 0.1 mol/L L-NAME to determine nonspecific activity. The reaction was stopped by placing the tubes on ice and adding 20 mmol/L ice-cold Hepes buffer (pH 5.5) followed by separation of arginine and citrulline on Dowex 50 x 8 to 200 (Na+ form). 3H-citulline was detected by scintillation counting. All measurements were performed in duplicate, and the results are expressed as picomoles per minute per milligram of protein.

Renal Cortex NOS Protein Mass
This assay was performed to determine the eNOS, inducible NOS (iNOS), and neuronal NOS protein mass as described previously (25). Anti-eNOS, anti-iNOS, and anti–neuronal NOS monoclonal antibodies and the respective positive controls as well as the peroxidase-conjugated goat anti-mouse IgG antibody were purchased from Transduction Laboratories. Briefly, 100 µg of kidney tissue preparations were size-fractionated on 4 to 12% Tris-Glycine gel (Novex) at 120 V for 3 h. After electrophoresis, proteins were transferred onto Hybond-ECL membrane (Amersham Life Science) at 400 mA for 120 min using the Novex transfer system. The membrane was prehybridized in 10 ml of buffer A (10 mmol/L Tris hydrochloride [pH 7.5], 100 mmol/L NaCl, 0.1% Tween 20, and 10% nonfat milk powder) for 1 h and then hybridized for an additional 1-h period in the same buffer containing 10 µl of the given anti-NOS monoclonal antibody (1:1000). The membrane was then washed for 30 min in a shaking bath, with the wash buffer (buffer A without nonfat milk) changed every 5 min before 1 h of incubation in buffer A plus goat anti-mouse IgG–horseradish peroxidase at the final titer of 1:1000. Experiments were carried out at room temperature. The washes were repeated before the membrane was developed with a light-emitting nonradioactive method using ECL reagent (Amersham). The membrane was then subjected to autoluminography for 1 to 5 min. The autoluminographs were scanned with a laser densitometer (model PD1211; Molecular Dynamics, Eugene, OR) to determine the relative optical densities of the bands. In all instances, the membranes were stained with Ponceau stain before hybridization. This step verified the uniformity of protein load and transfer efficiency across the test samples (26).

Renal Cortex Caveolin-1 Protein Mass
Kidney slices were ground to powder with a precooled pestle, in a mortar filled with a small amount of liquid nitrogen. After nitrogen was evaporated, samples were collected and homogenized in a glass Potter with 100 µl of buffer (composition: 20 mmol/L Tris-HCl [pH 7.4], 2.5 mmol/L EDTA, 100 mmol/L NaCl, 10 mmol/L NaF, 1 mmol/L Na3VO4, 1% NaDOC, 0.1% SDS, 1% Triton X-100, and a cocktail of protease inhibitors [Sigma]). Samples were then transferred into microtubes and centrifuged for 10 min at 10,000 x g, and the supernatants were collected for immunoblotting experiments. Protein samples (30 to 60 µg protein/lane) were subjected to electrophoresis on 10% SDS-PAGE gels, then transferred onto PVDF membranes. The membranes were incubated overnight in 5% low-fat milk in PBS-Tween buffer at 4°C. Immunoblotting was then performed as described previously (20).

Statistical Analyses
Results are expressed as mean ± SEM. Quantitative data were tested by two-way ANOVA. The Kruskal-Wallis ANOVA tested semiquantitative data. When the variance ratio (F) reached statistical significance (P < 0.05), quantitative data were analyzed further with the Student-Newman-Keuls test for multiple comparisons, and semiquantitative data were analyzed further with Dunn test for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protocol 1
Two weeks of cholesterol feeding significantly increased plasma cholesterol levels (Figure 1A) and glomerular lipid deposition (Figure 1B). Dietary cholesterol had no effect on SBP (Table 1). Glomerular sclerosis and interstitial fibrosis were not observed. Transmission electron microscopy of glomeruli of cholesterol-treated rats showed focal fusion of foot processes of epithelial cells of high electron density, as a result of condensed cytoskeleton (Figure 2A), which was practically absent in control rats (Figure 2B). Rats that were fed cholesterol had marked glomerular epithelial desmin staining, a marker of podocyte activation (Figure 2, C versus D), and a significant increase in glomerular edge desmin score (Figure 2E). Desmin staining was practically absent in most glomeruli of control rats (Figure 2D). Renal cortex NOS activity was significantly decreased (Figure 3A). However, renal cortical bNOS, eNOS, and iNOS protein masses were not significantly changed (Figure 3B). Cortex caveolin-1 protein mass was increased by nearly 40% in rats that were fed cholesterol (Figure 4). Glomerular caveolin, although clearly present in controls (Figure 5B), was significantly increased by cholesterol (Figure 5A), positive surface area being increased by nearly 50% (Figure 5C). Note that caveolin staining was not restricted to the visceral epithelium but also seemed to be present in endothelial and mesangial cells.



View larger version (29K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. Plasma cholesterol (A) and glomerular lipid deposits (B) in controls, rats that were fed 2% cholesterol for 2 wk, rats that were fed 2% cholesterol for 2 wk and were pretreated for 2 wk with molsidomine or losartan, and rats that received 40 mg of N-{omega}-nitro-L-arginine (L-NNA)/kg diet for 2 wk without or with pretreatment with losartan for 2 wk. *P < 0.05 versus control.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Effects of hypercholesterolemia without or with molsidomine or losartan treatment and L-NNA without or with losartan treatment on plasma creatinine, proteinuria, and SBP in female ratsa
 


View larger version (85K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. (A) In a cholesterol-treated rat, focal fusion foot processes of epithelial cells of high electron density, as a result of local accumulation of condensed cytoskeleton, can be observed. (B) Part of a glomerular loop of a control rat showing electron-dense foot processes with a low density of condensed cytoskeleton. Larger processes are also present. Bar = 1 µm. (C) Desmin in a glomerulus of a female rat with dietary hypercholesterolemia. (D) Desmin staining was virtually absent in the glomerulus of a control rat. (E) Desmin staining in controls, rats that were fed 2% cholesterol for 2 wk, rats that were fed 2% cholesterol for 2 wk and were pretreated for 2 wk with molsidomine or losartan, and rats that received 40 mg L-NNA/kg diet for 2 wk without or with pretreatment with losartan for 2 wk. *P < 0.05 versus control; {dagger}P < 0.05 versus 2% cholesterol; #P < 0.05 versus L-NNA.

 


View larger version (28K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. (A) Renal cortex nitric oxide synthase (NOS) activity in controls, rats that were fed 2% cholesterol for 2 wk, rats that were fed 2% cholesterol for 2 wk and were pretreated for 2 wk with molsidomine or losartan, and rats that received 40 mg of L-NNA/kg diet for 2 wk without or with pretreatment with losartan for 2 wk. *P < 0.05 versus control; {dagger}P < 0.05 versus 2% cholesterol. (B) Representative Western blot and data depicting NOS isoform protein abundance in renal cortex of control rats and rats that were fed 2% cholesterol for 2 wk.

 


View larger version (38K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. Representative Western blot and data depicting caveolin-1 protein abundance in renal cortex of control rats (lane 1), rats that were treated with 120 mg losartan/L drinking water for 4 wk (lane 2), rats that were fed 2% cholesterol for 2 wk (lane 3), and rats that were fed 2% cholesterol for 2 wk and were pretreated for 2 wk with losartan (lane 4). *P < 0.05 versus control; {dagger}P < 0.05 versus 2% cholesterol.

 


View larger version (96K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 5. Glomerular caveolin-1 staining in a cholesterol-fed rat (25% positive; A) and in a control rat (15% positive; B). (C) Data depicting percentage of caveolin-1 protein in glomeruli of control rats, rats that were treated with 120 mg losartan/L drinking water for 4 wk, rats that were fed 2% cholesterol for 2 wk, and rats that were fed 2% cholesterol for 2 wk and were pretreated for 2 wk with losartan. *P < 0.05 versus control.

 
Losartan had no influence on plasma cholesterol levels (Figure 1A) and on glomerular lipid deposits (Figure 1B) in cholesterol-fed rats and in rats that were fed normal diet (data not shown). SBP was slightly lower in all rats that received losartan (Table 1). Treatment with losartan prevented podocyte activation in cholesterol-fed rats (Figure 2E) and prevented the decrease of renal NOS activity (Figure 3A). Treatment with losartan decreased renal cortex caveolin-1 (Figure 4) and glomerular calveolin-1 (Figure 5) levels to normal. Losartan had no effect on renal cortex NOS activity (8.49 ± 0.53 versus 8.54 ± 0.38 pmol/min per mg protein in control) or glomerular caveolin-1 (Figure 5C) in rats that were fed a normal diet.

Molsidomine had no influence on plasma cholesterol levels (Figure 1A) and on glomerular lipid deposits (Figure 1B) in cholesterol-fed rats and in rats that were fed a normal diet (data not shown). Note that molsidomine also had no influence on SBP (Table 1). Treatment with molsidomine prevented podocyte activation in cholesterol-fed rats (Figure 2E) but also significantly decreased renal cortex NOS activity in rats that were fed cholesterol (Figure 3A) and in rats that were fed a normal diet (4.96 ± 0.26 versus 8.54 ± 0.38 pmol/min per mg protein in control; P < 0.05 versus control). None of these treatments had any effect on plasma creatinine (Table 1), triglycerides (data not shown), or proteinuria (Table 1).

Protocol 2
There were no significant effects of L-NNA on plasma cholesterol (Figure 1A), glomerular lipid deposits (Figure 1B), creatinine (Table 1), and plasma triglycerides (data not shown). L-NNA for 2 wk significantly increased SBP, and losartan treatment prevented this (Table 1). L-NNA, with or without 2 wk of pretreatment with losartan, had no influence on proteinuria. L-NNA–treated rats had significantly increased podocyte activation, which was prevented by losartan treatment (Figure 2E). L-NNA–treated rats had decreased renal cortex NOS activity, which was not corrected by losartan (Figure 3A).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main finding of this study is that hypercholesterolemia- or NOS inhibition–induced early podocyte stress is prevented by losartan before the onset of proteinuria. Molsidomine, an NO donor, prevented podocyte stress in hypercholesterolemic rats, suggesting that podocyte stress was due to NO deficiency. Furthermore, hypercholesterolemia decreased renal cortical NOS activity and increased renal cortical and glomerular caveolin protein, which were also prevented by losartan.

Despite a similar degree of hypercholesterolemia, podocyte injury was much less pronounced at this early stage than in our previous studies, in which after 3 mo hypercholesterolemia had lead to proteinuria (3,27). Transmission electron microscopy showed only occasional fusion of some epithelial foot processes with focal accumulation of condensed cytoskeleton. At a later stage when proteinuria was present, we previously observed pseudocyst formation and extensive foot process effacement (27). Nevertheless, these mild changes in morphology were accompanied by a clear increase in desmin staining, suggesting that hypercholesterolemia as such already leads to podocyte stress. This podocyte stress may have been associated with NO deficiency because decreased renal cortical NOS activity as a result of hypercholesterolemia or NOS inhibition accompanied podocyte stress. However, we must acknowledge that this association does not necessarily suggest causality and that changes in renal cortical NOS activity may not be reflected by parallel changes within the glomerulus.

Exogenous NO administration with molsidomine reduced podocyte stress. Molsidomine increases cellular levels of cGMP by stimulating soluble guanylate cyclase directly (4,28). The dose used protected hypercholesterolemic and uremic rats from developing proteinuria, and adverse effects were not reported (3,29). Podocytes do not express any of the isoforms of NOS. Whether podocytes are directly responsive to NO is debatable. Administration of NO to cultured glomerular epithelial cells increased production of cGMP, which controlled the cytoskeletal structure and limited retraction (14). However, it has recently been described that podocytes in situ do not express soluble guanylate cyclase (30). Mesangial cells express eNOS and iNOS (31) as well as soluble guanylate cyclase (30). Mesangial cell contractility is known to influence podocyte conformation (32) and hence also influences podocyte stress. Even though podocytes express LDL receptors and cultured glomerular epithelial cells bind and take up VLDL (33) and IDL (34), this is probably not relevant in the present study, because neither losartan nor molsidomine decreased plasma cholesterol levels in cholesterol-fed rats, but both prevented podocyte stress. Furthermore, glomerular lipid deposits in cholesterol-fed rats, determined by the Sudan black staining, were not altered by treatment with either losartan or molsidomine. Thus, early podocyte stress in hypercholesterolemia may be linked to NO deficiency rather than to cholesterol uptake.

In the kidney, the heart, and blood vessels, NO counteracts angiotensin II activity and is considered to be a safety factor (17–19). Hypercholesterolemia enhanced biologic activity of angiotensin II in experimental models (5–12) and in humans (35). It has been suggested that in the vasculature, angiotensin II increases superoxide production and lipid peroxidation, which decreases endothelial NO availability (19), and blockade of the renin angiotensin II system was shown to increase NOS activity in endothelial cells in aging rats (36). Treatment with AT-1 receptor blockers (10) and ACE inhibitors (37) prevented lipid peroxidation, increased endothelial NO availability, and prevented early hypercholesterolemia-induced atherogenesis. Furthermore, ACE inhibition improved endothelium-dependent relaxation in hypercholesterolemic patients (38). It has also been shown that losartan prevented renal injury during chronic NOS inhibition (18). In the current study, prevention of podocyte stress by losartan in the L-NNA rats suggests that when NO availability is low, angiotensin II may have a direct noxious effect on podocytes. Indeed, as discussed above, NO deficiency leads to podocyte stress. Podocytes are probably subject to opposing effects of angiotensin II and NO, as is the case for vascular smooth muscle cells. It has been shown that podocytes express AT-1 receptors (13), and stimulation with angiotensin II increased intracellular calcium influx and superoxide activity (39). Stimulation of cultured podocytes with angiotensin II increased cAMP levels and caused enhanced actin aggregation (14). We found previously that NOS inhibition, before the development of injury, lowers renal angiotensin II levels and that, nevertheless, losartan was an effective protective agent (18). In double-knockout eNOS- and apolipoprotein E–deficient mice, dietary hypercholesterolemia induced atherosclerosis and kidney dysfunction that were abrogated by an ACE inhibitor (40). Thus, the hypercholesterolemia-induced podocyte stress can be due to direct effects of angiotensin II, unopposed by endogenous NO. However, podocyte stress is often associated with glomerular hypertension (41), and we cannot exclude that the hypercholesterolemia induced glomerular hypertension (42). In addition, there was a slight decrease in SBP induced by losartan in the hypercholesterolemic rats (117 versus 128 mmHg), and in the L-NNA–treated rats, the antihypertensive effect of losartan was stronger (110 versus 138 mmHg). Thus, in the cholesterol plus losartan and L-NNA plus losartan groups, reduction of podocyte stress by a hemodynamic component is possible. However, it should be noted that molsidomine prevented podocyte stress without affecting BP.

Hypercholesterolemia decreased renal cortical NOS activity and increased cortical and glomerular caveolin protein, and this was prevented by losartan. The upregulation of renal cortical caveolin-1 may account for decreased renal cortical NOS activity, analogous to what we have observed in endothelial cells (20). However, it should be noted that we did not measure cortical NOS activity in a single compartment. The caveolin–NOS interaction maybe angiotensin II sensitive, because losartan treatment prevented the increase in caveolin-1 protein abundance and decrease in renal cortex NOS activity induced by hypercholesterolemia. Caveolin-1 binds to eNOS, thereby inhibiting its activity and decreasing NO availability. Thus, if caveolin-1 is upregulated, then more calmodulin is required to destabilize the inhibitory caveolin/eNOS complex. Lack of caveolin-1 expression, both in kidney cells and in mice, prevents trafficking of the AT-1 receptor from the endoplasmic reticulum to the cell surface (43). The present study has, to our knowledge, for the first time demonstrated hypercholesterolemia-induced increase of renal cortical and, more specific, glomerular, caveolin protein abundance and decrease of renal cortical NOS activity and that these changes may be angiotensin II sensitive. Losartan treatment alone had no effect on renal cortical and glomerular caveolin, suggesting a low baseline activity of angiotensin II in the kidney. In vascular smooth cells, angiotensin II was found to increase caveolin-1 biosynthesis (21), and in cultured endothelial cells, angiotensin II was found to decrease NOS activity (22). After 2 wk of hypercholesterolemia, the protein abundance of all NOS isoforms in the renal cortex was unchanged (this study), and previously we also found that morphometric quantification of glomerular eNOS was unaltered (3). Thus, it is possible that hypercholesterolemia decreased renal cortical NOS activity by increasing caveolin-1 protein abundance via an AT-1–sensitive mechanism. However, although podocyte stress was also prevented by losartan in L-NNA–treated rats, it is unlikely that losartan could restore L-NNA–induced NO deficiency. Hence, even though in hypercholesterolemia losartan improved renal cortical NOS activity and decreased caveolin-1 protein mass and molsidomine and losartan were equally capable of preventing podocyte stress in cholesterol-fed rats, increasing NO availability is probably not the only protective action of AT-1 blockade.

An alternative candidate pathway involved in podocyte stress that might be targeted by AT-1 blockade is oxidative stress. In a previous study, we found that high-dose cholesterol feeding for a longer period increased renal superoxide activity in the presence of proteinuria (3). Similarly, in the porcine renal vasculature, chronic hypercholesterolemia increased oxidative stress (2). The same authors recently showed that chronic antioxidant supplementation preserved renal vascular function in hypercholesterolemia (44). However, in our model of short-term hypercholesterolemia, proteinuria and renal superoxide activity were not increased (3), which makes it unlikely that these targets were involved in the protective effect of losartan observed at this early stage.

In summary, the present study demonstrated that hypercholesterolemia increased podocyte stress, which was associated with NO deficiency and angiotensin II sensitivity. All of these events were early and occurred without proteinuria. We suggest that the decreased renal NO availability induced by hypercholesterolemia may leave the hemodynamic or direct effects of angiotensin II on podocytes unopposed. Furthermore, hypercholesterolemia decreased renal cortical NOS activity and increased renal cortical and glomerular caveolin protein abundance. Caveolin-1 protein abundance seemed to be increased via an angiotensin II–sensitive mechanism. It is interesting that in the present study, angiotensin II receptor blockade was applied before the onset of proteinuria. Proteinuria has been established as a risk factor for renal injury, and clinical trials have used AT-1 receptor blockade (45) and ACE inhibition (46) to reduce proteinuria and stabilize the rate of progression of renal injury. The clinical relevance of this study may be that in patients without proteinuria but with other risk factors for renal injury, early treatment with an AT-1 blocker or an ACE inhibitor might serve to prevent podocyte stress and the onset of proteinuria.


   Acknowledgments
 
The Dutch Kidney Foundation supported this study (grant C96.1608). Hennie IJzerman, Dionne van der Giezen, Paula Martens, and Zhenmin Ni provided expert technical assistance. We gratefully acknowledge their contribution to this study. Publication of Figures 2 and 5 Go in color is made possible by Merck Sharp & Dohme BV, Haarlem, the Netherlands.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Feldstein A, Krier JD, Hershman Sarafov M, Lerman A, Best PJM, Wilson SH, Lerman LO: In vivo renal vascular and tubular function in experimental hypercholesterolemia. Hypertension 34: 859–864, 1999[Abstract/Free Full Text]
  2. Stulak JM, Lerman A, Caccitolo JA, Wilson SH, Romero JC, Schaff HV, Rodriguez Porcel M, Lerman LO: Impaired renal vascular endothelial function in vitro in experimental hypercholesterolemia. Atherosclerosis 154: 195–201, 2001[CrossRef][Medline]
  3. Attia DM, Ni Z, Boer P, Attia MA, Goldschmeding R, Koomans HA, Vaziri ND, Joles JA: Proteinuria is preceded by decreased NO synthesis and prevented by a NO donor in cholesterol-fed rats. Kidney Int 61: 1776–1787, 2002[CrossRef][Medline]
  4. Feelisch M: The use of nitric oxide donors in pharmacological studies. Naunyn Schmiedebergs Arch Pharmacol 358: 113–122, 1998[CrossRef][Medline]
  5. Hayek T, Attias J, Smith J, Breslow JL, Keidar S: Antiatherosclerotic and antioxidative effects of captopril in apolipoprotein E-deficient mice. J Cardiovasc Pharmacol 31: 540–544, 1998[CrossRef][Medline]
  6. Keidar S, Attias J, Smith J, Breslow JL, Hayek T: The angiotensin-II receptor antagonist, losartan, inhibits LDL lipid peroxidation and atherosclerosis in apolipoprotein E-deficient mice. Biochem Biophys Res Commun 30: 622–625, 1997
  7. Schwemmer M, Sommer O, Bassenge E: Blockade of angiotensin signaling improves myocardial function in hypercholesterolemia independent of changes in eicosanoid release. Cardiovasc Drugs Ther 14: 317–327, 2000[CrossRef][Medline]
  8. Chen H, Li D, Sawamura T, Inoue K, Mehta JL: Upregulation of LOX-1 expression in aorta of hypercholesterolemic rabbits: Modulation by losartan. Biochem Biophys Res Commun 276: 1100–1104, 2000[CrossRef][Medline]
  9. Jacobsson LS, Persson K, Aberg G, Andersson RG, Karlber BE, Olsson AG: Antiatherosclerotic effects of angiotensin-converting enzyme inhibitors captopril and fosinopril in hypercholesterolemic minipigs. J Cardiovasc Pharmacol 24: 670–677, 1994[Medline]
  10. Strawn WB, Chappell MC, Dean RH, Kivlighn S, Ferrario CM: Inhibition of early atherogenesis by losartan in monkeys with diet-induced hypercholesterolemia. Circulation 101: 1586–1593, 2000[Abstract/Free Full Text]
  11. Atarashi K, Takagi M, Minami M, Ishiyama A: Effects of alacepril and amlodipine on the renal injury induced by a high-cholesterol diet in rats. J Hypertens 17: 1983–1986, 1999[CrossRef][Medline]
  12. Sakemi T, Baba N: Effects of antihypertensive drugs on the progress of renal failure in hyperlipidemic Imai rats. Nephron 63: 323–329, 1993[Medline]
  13. Nitschke R, Henger A, Ricken S, Muller V, Knoogen M, Bek M, Pavenstädt H: Angiotensin II increases the intracellular calcium activity in podocytes of the intact glomerulus. Kidney Int 57: 41–49, 2000[CrossRef][Medline]
  14. Sharma R, Lovell HB, Wiegmann TB, Savin VJ: Vasoactive substances induce cytoskeletal changes in cultured rat glomerular epithelial cells. J Am Soc Nephrol 3: 1131–1138, 1992[Abstract]
  15. Johnson RJ, Alpers CE, Yoshimura A, Lombardi D, Pritzl P, Floege J, Schwartz SM: Renal injury from angiotensin II-mediated hypertension. Hypertension 19: 464–474, 1992[Abstract/Free Full Text]
  16. Lombardi DM, Viswanathan M, Vio CP, Saveedra JM, Schwartz SM, Johnson RJ: Renal and vascular injury induced by exogenous angiotensin II is AT1 receptor-dependent. Nephron 87: 66–74, 2001[CrossRef][Medline]
  17. Zatz R, and Baylis C: Chronic nitric oxide inhibition model six years on. Hypertension 32: 958–964, 1998[Free Full Text]
  18. Verhagen MG, Braam B, Boer P, Gröne H-J, Koomans HA, Joles JA: Losartan-sensitive renal damage caused by chronic nitric oxide synthase inhibition does not involve increased renal angiotensin II concentrations. Kidney Int 56: 222–231, 1999[Medline]
  19. Dzau VJ: Theodore Cooper Lecture: Tissue angiotensin and pathobiology of vascular disease: A unifying hypothesis. Hypertension 37: 1047–1052, 2001[Abstract/Free Full Text]
  20. Feron O, Dessy C, Moniotte S, Desager J-P, Balligand J-L: Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase. J Clin Invest 103: 897–905, 1999[Medline]
  21. Ishizaka N, Griendling KK, Lassegue B, Alexander RW: Angiotensin II type 1 receptor: Relationship with caveolae and caveolin after initial agonist stimulation. Hypertension 32: 459–466, 1998[Abstract/Free Full Text]
  22. Li D, Tomson K, Yang B, Mehta P, Croker BP, Mehta JL: Modulation of constitutive nitric oxide synthase, bcl-2 and Fas expression in cultured human coronary endothelial cell exposed to anoxia-reoxygenation and angiotensin II: Role of AT1 receptor activation. Cardiovasc Res 41: 109–115, 1999[Abstract/Free Full Text]
  23. Floege J, Hackmann B, Kliem V, Kriz W, Alpers CE, Johnson RJ, Kühn KW, Koch K-M, Brunkhorst R: Age-related glomerulosclerosis and interstitial fibrosis in Milan normotensive rats: A podocyte disease. Kidney Int 51: 230–243, 1997[Medline]
  24. Sato T, Shamoto M: A simple rapid polychrome stain for epoxy-embedded tissue. Stain Technol 48: 223–227, 1973[Medline]
  25. Vaziri ND, Ni Z, Oveisi F: Upregulation of renal and vascular nitric oxide synthase in young spontaneously hypertensive rats. Hypertension 31: 1248–1254, 1998[Abstract/Free Full Text]
  26. Vaziri ND, Ding Y, Ni Z: Compensatory upregulation of nitric oxide synthase isoforms in lead-induced hypertension; reversal by a superoxide dismutase-mimetic drug. J Pharmacol Exp Ther 298: 679–685, 2001[Abstract/Free Full Text]
  27. Joles JA, Kunter U, Janssen U, Kriz W, Rabelink TJ, Koomans HA, Floege J: Early mechanisms of renal injury in hypercholesterolemic or hypertriglyceridemic rats. J Am Soc Nephrol 11: 669–683, 2000[Abstract/Free Full Text]
  28. Kukovetz WR, Holzmann S: Mechanism of vasodilation by molsidomine. Am Heart J 190: 637–6401, 1985
  29. Benigni A, Zoja C, Noris M, Corna D, Benedetti G, Bruzzi I, Todeschini M, Remuzzi G: Renoprotection by nitric oxide donor and lisinopril in the remnant kidney model. Am J Kidney Dis 33: 746–753, 1999[Medline]
  30. Theilig F, Bostanjoglo M, Pavenstädt H, Grupp C, Holland G, Slosarek I, Gressner AM, Russwurm M, Koesling D, Bachmann S: Cellular distribution and function of soluble guanylyl cyclase in rat kidney and liver. J Am Soc Nephrol 12: 2209–2220, 2001[Abstract/Free Full Text]
  31. Sandau KB, Brune B: Molecular actions of nitric oxide in mesangial cells. Histol Histopathol 15: 1151–1158, 2000[Medline]
  32. Kriz W, Hackenthal E, Nobiling R, Sakai T, Elger M: A role for podocytes to counteract capillary wall distension. Kidney Int 45: 369–376, 1994[Medline]
  33. Gröne H-J, Walli AK, Gröne E, Krämer A, Clemens MR, Seidel D: Receptor mediated uptake of apo B and apo E rich lipoproteins by human glomerular epithelial cells. Kidney Int 37: 1449–1459, 1990[Medline]
  34. Krämer A, Nauck M, Pavenstädt H, Schwedler S, Wieland H, Schollmeyer P, Wanner C: Receptor-mediated uptake of IDL and LDL from nephrotic patients by glomerular epithelial cells. Kidney Int 44: 1341–1351, 1993[Medline]
  35. Nickenig G, Wassmann S, Bohm M: Regulation of the angiotensin AT1 receptor by hypercholesterolemia. Diabetes Obes Metab 2: 223–228, 2000[CrossRef][Medline]
  36. González Bosc LV, Kurnjek ML, Müller A, Terragno NA, Basso N: Effect of chronic angiotensin II inhibition on the nitric oxide synthase in the normal rat during aging. J Hypertens 19: 1403–1409, 2001[CrossRef][Medline]
  37. Napoli C, Cicala C, Darmiento FP, Roviezzo F, Somma P, de Nigris F, Zuliani P, Bucci M, Aleotti L, Casini A, Franconi F, Cirino G: Beneficial effects of ACE-inhibition with zofenopril on plaque formation and low-density lipoprotein oxidation in Watanabe heritable hyperlipidemic rabbits. Gen Pharmacol 33: 467–477, 1999[CrossRef][Medline]
  38. Esper RJ, Machado R, Vilarino J, Cacharron JL, Ingino CA, Garcia Guinazau CA, Bereziuk E, Bolano AL, Suarez DH: Endothelium-dependent responses in patients with hypercholesterolemic coronary disease under the effects of simvastatin and enalapril, either separately or combined. Am Heart J 140: 684–689, 2000[CrossRef][Medline]
  39. Greiber S, Münzel T, Kästner S, Müller B, Schollmeyer P, Pavenstädt H: NAD(P)H oxidase activity in cultured human podocytes: Effects of adenosine triphosphate. Kidney Int 53: 654–663, 1998[CrossRef][Medline]
  40. Kriz W, Gretz N, Lemley KV: Progression of glomerular diseases: Is the podocyte the culprit? Kidney Int 54: 687–697, 1998[CrossRef][Medline]
  41. Knowles JW, Reddick RL, Jennette JC, Shesely EG, Smithies O, Maeda N: Enhanced atherosclerosis and kidney dysfunction in eNOS-/- Apoe-/- mice are ameliorated by enalapril treatment. J Clin Invest 105: 451–458, 2000[Medline]
  42. Kasiske BL, O’Donnell MP, Schnitz PG, Kim Y, Keane WF: Renal injury of diet-induced hypercholesterolemia in rats. Kidney Int 37: 880–891, 1990[Medline]
  43. Wyse BD, Prior IA, Qian H, Morrow IC, Nixon S, Muncke C, Kurzchalia TV, Thomas WG, Parton RG, Hancock JF: Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane. J Biol Chem 278: 23738–23746, 2003[Abstract/Free Full Text]
  44. Stulak JM, Lerman A, Porcel MR, Caccitolo JA, Romero JC, Schaff HV, Napoli C, Lerman LO: Renal vascular function is preserved by chronic antioxidant supplementation. J Am Soc Nephrol 12: 1882–1891, 2001[Abstract/Free Full Text]
  45. Brenner BM, Cooper ME, De Zeeuw D, Keane WF, Mitch WE, Parving HH, Remuzzi G, Snapinn SM, Zhang Z, Shahinfar S: Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med 345: 861–869, 2001[Abstract/Free Full Text]
  46. Lewis EJ, Hunsicker LG, Bain RP, Rohde RD: The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy. N Engl J Med 329: 1456–1462, 1993[Abstract/Free Full Text]
Received for publication December 20, 2001. Accepted for publication December 21, 2003.




This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
S. Racasan, B. Braam, H. A. Koomans, and J. A. Joles
Programming blood pressure in adult SHR by shifting perinatal balance of NO and reactive oxygen species toward NO: the inverted Barker phenomenon
Am J Physiol Renal Physiol, April 1, 2005; 288(4): F626 - F636.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Attia, D. M.
Right arrow Articles by Joles, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Attia, D. M.
Right arrow Articles by Joles, J. A.

HOME CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP