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Am J Physiol Renal Physiol 294: F628-F637, 2008. First published December 19, 2007; doi:10.1152/ajprenal.00524.2007
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Eplerenone potentiates the antiproteinuric effects of enalapril in experimental nephrotic syndrome

Farid Nakhoul,1,* Eliyahu Khankin,1,6,* Afif Yaccob,2 Hiroshi Kawachi,3 Tony Karram,4 Huda Awaad,2 Nakhoul Nakhoul,5 Aaron Hoffman,4 and Zaid Abassi2,4

1Ambulatory Nephrology Unit and 4Department of Vascular Surgery, Rambam-Health Care Campus, and 2Department of Physiology and Biophysics, Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel; 3Department of Cell Biology, Institute of Nephrology, Niigata, University Graduate School of Medical and Dental Sciences, Niigata, Japan; 5Faculty of Medicine, Semmelweis University, Budapest, Hungary; and 6Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts

Submitted 7 November 2007 ; accepted in final form 13 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nephrotic syndrome (NS) is a clinical state characterized by massive proteinuria and edema. It is believed that nephrin and podocin are involved in the development of proteinuria. The proteinuria and effects of eplerenone alone or combined with enalapril on nephrin/podocin abundance in rats with NS have not yet been studied. Therefore, the present study was designed to examine the early (beginning 2 days before NS induction) and late (beginning 2 wk after NS induction) effects of eplerenone and enalapril, alone or combined, on proteinuria and nephrin/podocin abundance in rats with adriamycin-induced NS. Adriamycin caused a significant increase in daily protein excretion (UprV; from 26.96 ± 3.43 to 958.57 ± 56.7 mg/day, P < 0.001) and cumulative proteinuria [from 900.33 ± 135.5 to 22,490.62 ± 931.26 mg (P < 0.001)] during 6 wk. Early treatment with enalapril significantly decreased UprV from 958.6 ± 56.7 to 600.31 ± 65.13 mg/day (P < 0.001) and cumulative proteinuria to 12,842.37 ± 1,798.17 mg/6 wk (P < 0.001). Similarly, early treatment with eplerenone produced a profound antiproteinuric effect: UprV decreased from 958.57 ± 56.7 to 593.38 ± 21.83 mg/day, P < 0.001, and cumulative proteinuria to 16,601.84 ± 1,334.31 mg/6 wk; P < 0.001. An additive effect was obtained when enalapril and eplerenone were combined: UprV decreased from 958.57 ± 56.69 to 424.17 ± 38.54 mg/day, P < 0.001, and cumulative protein excretion declined to 10,252.88 ± 1,011.3 mg/6 wk, P < 0.001. These antiproteinuric effects were associated with substantial preservation of glomerular nephrin and podocin. In contrast, late treatment with either enalapril or eplerenone alone or combined mildly decreased UprV and cumulative proteinuria. Thus pretreatment with eplerenone or enalapril is effective in reducing daily and cumulative protein excretion and preservation of nephrin/podocin. More profound antiproteinuric effects were obtained when enalapril and eplerenone were combined.

adriamycin; proteinuria; nephrin; podocin

NEPHROTIC SYNDROME (NS) is one of the most severe and debilitating diseases encountered in nephrology. Clinically, it is characterized by massive proteinuria, hypoalbuminemia, edema state, and hyperlipidemia. It is well known that the protein barrier of the glomerular membrane is charge selective for smaller proteins and size selective for larger proteins (9, 19, 22). The slit diaphragm, spanning the area between two adjacent podocyte foot processes, has an important and direct role in glomerular filtration and selectivity (8, 12). It has been well established that its protein components are involved in the mechanism of proteinuria development. Nephrin and podocin are two of the slit diaphragm structural proteins and are essential for maintaining a functional glomerular filtration barrier (12, 20, 22). Mutations in the nephrin gene (NPHS1) were identified in 90% of patients with congenital NS of the Finnish type (23, 53), and in 50% of patients with no Finnish ancestry, resulting in massive proteinuria already in utero (27, 30, 35, 37). Podocin is a membrane-associated protein, which interacts with the cytosolic tail of nephrin, (8, 48). In fact, presence of intact podocin is a prerequisite for the targeting of nephrin to the podocyte membrane and for podocyte intracellular signaling. Mutations in the podocin gene (NPHS2) cause severe structural podocyte alterations and massive proteinuria leading to nephrotic syndrome (6, 28, 43, 48, 50).

The adriamycin (ADR)-induced NS model in the rat is characterized by massive proteinuria, hypoalbuminemia, and edema formation (4). The main feature of this experimental nephropathy, which mimics to some extent minimal-change NS as well as focal segmental glomeruloscelrosis, is glomerular dysfunction resulting in a massive proteinuria.

The renin-angiotensin-aldosterone system (RAAS) is well known to be involved in the development of proteinuria in a variety of clinical and experimental glomerular diseases (2, 21). Beneficial effects of early treatment with various antiproteinuric therapies, such angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARBs), on proteinuria and glomerular abundance of nephrin and podocin were demonstrated by many groups including ours (3, 7, 26, 29, 33, 38). Combination therapy (most frequently combining ACEI and ARB) is frequently used to achieve improved antiproteinuric effects (11, 24, 51, 52). However, the effectiveness of such a combination in reducing proteinuria compared with monotherapy is still far from being resolved (31, 51, 52).

In recent years, there has been a paradigm shift with respect to our understanding of aldosterone's widespread effects on the blood vessels in general, with special focus on vasculature of heart and kidney. There is an increasing body of evidence that aldosterone has an effect on vasculature remodeling and collagen formation and has an epigenomic capacity to modify endothelial function (1, 10, 15, 34, 40). These effects contribute substantially to the pathogenesis of congestive heart failure, as well as progressive renal dysfunction (1, 34, 40). Naturally, these observations led to an appreciation of the need for developing selective antagonists blocking aldosterone receptors, having some diuretic effect, but primarily targeting a potential cardioprotective as well and renoprotective effect (1, 13, 14, 36, 44).

Growing evidence suggests that aldosterone contributes to the development of proteinuria. Aldosterone has been linked to the progression of renal disease independently of its hypertensive effects (10, 40). The mechanism responsible for the progression of aldosterone-induced renal injury was investigated in different animal models. Nishiyama and Abe (34) demonstrated that chronic administration of aldosterone in rats results in severe proteinuria and glomerular injury, characterized by cell proliferation and mesangial matrix expansion. Increased renal cortical NAD(P)H oxidase expression, reactive oxygen species generation, and mitogen-activated protein kinase activation were also observed (34). Treatment with eplerenone, a selective aldosterone antagonist, prevented these changes and ameliorated glomerular injury (18, 45, 56). Sato et al. (46) and others (18, 47) have recently shown that mineralocorticoid receptor blockade (MRB) may represent optimal therapy for patients suffering from diabetic nephropathy associated with chronic renal disease and proteinuria. They suggested that attenuation of MR-mediated effects by an aldosterone antagonist may become a new goal for patients who escape the antiproteinuric effects of conventional treatments. Unfortunately, no studies testing the effectiveness of eplerenone alone or in combination with ACEI as treatment for proteinuria in an experimental model of ADR-induced NS in rats are available.

Therefore, the present study was designed to examine the antiproteinuric effect of eplerenone as well as to evaluate whether concomitant administration of eplerenone with ACE-I offers an antiproteinuric effect superior to that of each compound alone.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Studies were conducted in male Sprague-Dawley rats (Harlan Laboratories, Jerusalem, Israel), weighing ~330 g. Animals were kept in individual metabolic cages in a temperature-controlled room and fed standard rat chow, containing 0.5% NaCl and tap water ad libitum. All experiments were performed according to the guidelines of the Committee for the Supervision of Animal Experiments, Technion-Israel Institute of Technology.

Experimental Model

NS was induced by a single dose of ADR (5 mg/kg body wt) injected into the tail vein of conscious rats (n = 9, and n = 11, in early and late treatment groups, respectively) according to the protocol of Bertani et al. (4). Animals injected with vehicle only served as controls (n = 8). Previously, our group (33) demonstrated that ADR-induced NS in the rat is characterized by massive proteinuria, hypoalbuminemia, edema, and ascites formation. Established proteinuria was observed 2 wk following the ADR administration, resulting in death within 6 wk. Postmortem examination revealed extensive edema, ascites, lung congestion, and pleural effusion. Therefore, pharmacological intervention in treatment protocols was limited to 5 wk. Animals from the various study groups survived the study.

Treatment Protocols

Protocols were designed to evaluate the antiproteinuric effect of long-term early administration (5 wk) of enalapril (Sigma, St. Louis, MO), an ACEI, and eplerenone, an aldosterone antagonist (Pfizer), alone or in combination. Enalapril was given in drinking water (100 mg/l). Eplerenone was mixed in chow (120 mg/100 g). Urinary protein excretion (UprV), sodium excretion (UNaV), plasma levels of albumin, total proteins, and lipids were determined. Food intake was adjusted every 3 days to maintain the dose of eplerenone as targeted by the study design.

Rats were housed in individual metabolic cages for 1 wk to obtain baseline values of urinary protein and sodium excretion. During this period, mean arterial blood pressure (MAP) was measured using a tail-cuff method (IITC, model 31, Woodland Hills, CA) after the animals were maintained in an incubator at 37°C for 15 min to ensure vasodilatation. For each rat, MAP was measured at least three times. Rats were randomized to receive enalapril or eplerenone either 2 days before ADR injection (early treatment) or 2 wk after ADR (late treatment). A combination of enalapril and eplerenone at the above-mentioned doses was administered as described above. Efficacy of the treatment was monitored by daily measurements of UprV and UNaV for 5 wk.

Nephrotic rats that received no treatment served as controls. Enalapril dosing is based on protocols described by Taal and Brenner (51, 52); eplerenone dosing is based on a protocol published by Zhou et al. (56). Rats drink ~20–25 ml of water/day, resulting in a dose of 7–9 mg·kg–1·day–1 of enalapril, and eat 25 g, resulting in a dose of 100 mg·kg–1·day–1 of eplerenone (44). Exactly 6 wk after the beginning of the study, rats from different experimental group were anesthetized (30 mg/kg ip pentobarbital sodium), a tracheostomy was performed, and polyethylene catheters (PE-50) were inserted into the left carotid artery for measurements of arterial blood pressure and collection of blood samples. Kidneys were perfused with normal saline and harvested for immunohistochemical determination of nephrin and podocin.

Immunohistochemistry

Whole kidneys were rapidly frozen in liquid nitrogen, and 4-µm-thick cryostat sections were placed on Silan-coated slides and dried at room temperature (RT). Sections were fixed in acetone/ethanol (4:1) solution for 10 min and washed in PBS. Sections were incubated with 10% normal goat serum (NGS) in PBS at RT for 1 h, followed by a 2-h incubation at RT with an anti-nephrin antibody (polyclonal antibody) or an anti-podocin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:200 in PBS containing 1% NGS. Slides were then washed in PBS and incubated for 1 h at RT with FITC-conjugated goat anti-rabbit IgG (Chemicon, Temecula, CA) diluted in PBS containing 1% NGS. Nephrin and podocin immunoreactivities were analyzed by measuring fluorescence intensity, utilizing a digital image analyzer (Image Pro-Plus, Media Cybernetics) and a low-light video camera (MicroMax 1300Y).

Chemical Analysis

Urinary protein concentration was determined by using spectrophotometry, after 3% sulfosalicylic acid precipitation of urine collected from rats individually housed in metabolic cages for 24 h throughout the experimental period. Concentration of creatinine in plasma and in urine was measured by the colorimetric anthrone method. Plasma albumin and proteins concentrations were determined using a refractometer (Biochemical Laboratories, Rambam Medical Center, Haifa, Israel). Sodium and potassium concentrations in plasma and urine were determined by flame photometry (model IL 943, Instrumentation Laboratories).

Statistical Analysis

One-way ANOVA followed by the Dunnett test or two-way ANOVA was used. A P value <0.05 was considered statistically significant. Data are presented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Chronic Administration of Various Drugs

Daily and cumulative protein excretion. In line with our previous reports (33), animals treated with ADR exhibited a rapid increase in absolute and cumulative protein excretion. Absolute urinary protein excretion increased from basal values of 26.96 ± 3.43 to 958.57 ± 56.69 mg/day (P < 0.001) 6 wk after ADR administration (Fig. 1). Since urinary protein excretion in nephrotic rats shows daily fluctuations, we also present cumulative data on the antiproteinuric effects of the various treatments. As shown in Fig. 1, cumulative protein excretion was very low in healthy controls, 900.33 ± 135.53 mg during 6 wk of follow-up, and increased to 22,490.62 ± 931.26 mg during the same period in untreated NS animals (P < 0.001). Early treatment with enalapril caused a significant decrease in daily and cumulative proteinuria: Absolute urinary protein excretion decreased by 35% in enalapril-treated NS animals, at week 6 (from 958.57 ± 56.69 to 600.3 ± 65.13 mg/day P < 0.001) (Fig. 1A). Cumulative protein excretion in untreated and enalapril-treated NS rats is depicted in Fig. 1. Two-way ANOVA clearly shows a profoundly significant (P < 0.001) reduction in the cumulative protein excretion in NS rats treated with enalapril early (12,842.3 ± 1,798.17 mg), compared with untreated nephrotic rats (22,490.62 ± 931.26 mg). In contrast, absolute daily urinary protein excretion decreased by 20% in NS animal at week 6 (from 709.5 ± 32.63 to 564.62 ± 98.4 mg/day, P = 0.14) when enalapril was given late (Fig. 1B). Cumulative protein excretion values of both untreated and late enalapril-treated NS rats are depicted in the same figure. Two-way ANOVA clearly shows no significant reduction (P > 0.05) in the cumulative protein excretion in NS rats that were treated with enalapril late (19,903.9 ± 1,940.8 mg) compared with untreated nephrotic rats.


Figure 1
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  		Fig. 1. Effects of chronic administration of enalapril given via drinking water either as early (n = 7; A) or as late therapy (n = 5; B) on daily protein excretion and cumulative protein excretion in rats with nephrotic syndrome (NS) compared with untreated NS animals (n = 9 and n = 11 in early and late treatment groups, respectively). *P < 0.05 vs. baseline. #P < 0.05 vs. untreated NS rats.

 
Similar to enalapril, early treatment with eplerenone caused a significant decrease in daily and cumulative protein excretion beginning at week 4 of treatment and lasting throughout the experimental period (Fig. 2A). Absolute urinary protein excretion at week 6 decreased by 38% in early eplerenone-treated NS animals (from 958.57 ± 56.69 to 593.38 ± 21.83 mg/day P < 0.001) (Fig. 2). Cumulative urinary protein excretion values during 6 wk of follow-up of both untreated and eplerenone-treated NS rats are depicted in Fig. 2. There was a significant (P < 0.001, by ANOVA 2) reduction in the cumulative protein excretion in NS rats treated with eplerenone early (16,601.8 ± 1,334.3 mg) compared with untreated nephrotic rats (22,490.62 ± 931.26 mg). Late treatment with eplerenone did not decrease the daily or cumulative urinary protein excretion (daily urinary protein excretion was 709.5 ± 32.63 mg/day in untreated animals and 697.47 ± 32.03 mg/day in late-treated group P = 0.8445) (Fig. 2B).


Figure 2
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  		Fig. 2. Effects of chronic administration of eplerenone given via food either as early (n = 5; A) or as late therapy (n = 6; B) on daily protein excretion and cumulative protein excretion in rats with NS compared with untreated NS animals (n = 9 and n = 11 in early and late treatment groups, respectively). *P < 0.05 vs. baseline. #P < 0.05 vs. untreated NS rats.

 
Early administration of combination therapy caused a significant decrease in daily urinary protein excretion, which was more profound than that observed with early treatment using either drug alone (from 958.57 ± 56.69 to 424.17 ± 38.54 mg/day, P < 0.001) (Fig. 3A). Moreover, cumulative proteinuria decreased from 22,490.62 ± 931.26 to 10,252.88 ± 1,011.31 mg/day (P < 0.001). In contrast, late administration of combined enalapril and eplerenone caused a milder, yet still significant decrease in daily proteinuria at the 6-wk time point (from 709.5 ± 32.63 to 519.6 ± 30.4 mg/day P < 0.01) and cumulative proteinuria (from 21,057.27 ± 708.86 to 17,044.36 ± 1,026.22 mg/day, P < 0.001) (Fig. 3B).


Figure 3
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  		Fig. 3. Effects of chronic coadministration of enalapril and eplerenone given via drinking water and food, respectively, as either early or late (n = 5) therapy on daily protein excretion (A) and cumulative protein excretion (B) in rats with NS (n = 9 and n = 11 in early and late treatment groups, respectively) compared with untreated NS animals. *P < 0.05 vs. baseline. #P < 0.05 vs. untreated NS rats.

 
Normalization of urinary protein to urinary creatinine (Upr/Ucr) is presented in Fig. 4. Figure 4, A and B, show that rats with NS have higher Upr/Ucr compared with healthy controls (P < 0.001). Unexpectedly, Upr/Ucr increased following early enalapril treatment and decreased when the drug was given late. This trend is in line with our findings that the proportional decrease in Ucr was greater than for Upr in early enalapril-treated NS animals (data not shown). It should be emphasized that treatment with enalapril prevented weight loss that was observed in untreated NS animals, suggesting an improvement in their catabolic state and possibly a reduction of creatinine production. Eplerenone slightly and not significantly decreased Upr/Ucr in both early- and late-treated animals. The combination of eplerenone and enalapril decreased Upr/Ucr by 83 (P < 0.001) and 81% (P < 0.001) when the drugs were given early or late, respectively.


Figure 4
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  		Fig. 4. Effects of chronic administration of enalapril and eplerenone given either separately or in combination (n = 5–7) given as early (A) or late (B) therapy on daily urinary protein excretion normalized to daily urinary creatinine excretion (Uprot/Ucr) in rats with NS. *P < 0.05 vs. baseline. #P < 0.05 vs. untreated NS rats.

 
Albumin and total protein. As shown in Fig. 5, total plasma protein and albumin concentrations were significantly lower in untreated NS animals (4.56 ± 0.16 and 1.07 ± 0.14 g/dl respectively) compared with controls (5.42 ± 0.07 and 3.35 ± 0.11 g/dl, P < 0.05, respectively). Despite the antiproteinuric effect of enalapril, total plasma protein and albumin levels were not improved following drug administration, and both parameters were persistently low and comparable to their levels in untreated NS rats (Fig. 5). Early, and to a lesser extent, late treatment with eplerenone alone increased plasma levels of total protein and restored it to normal values (5.26 ± 0.26 and 5.08 ± 0.21 g/dl in early and late treatment, respectively, P > 0.05). Plasma albumin levels increased slightly and not significantly. Combined early and late treatment abolished the NS-associated decrease in plasma total protein (5.4 ± 0.03 and 5.42 ± 0.08 g/dl, P < 0.05, respectively) and improved plasma albumin levels only when given as early treatment (2.16 ± 0.12 g/dl, P > 0.05).


Figure 5
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  		Fig. 5. Effects of chronic administration of enalapril and eplerenone alone or in combination (n = 5–7) given as early or late therapy on total plasma protein (top) and plasma albumin levels (bottom) in rats with NS (n = 9 and n = 11 in early and late treatment groups, respectively). *P < 0.05 vs. baseline. #P < 0.05 vs. untreated NS rats.

 
Blood pressure. As shown in Fig. 6, MAP (measured via the carotid artery at week 6) in untreated NS rats was significantly lower compared with sham-treated controls (115.8 ± 2.2 vs. 129.7 ± 4.7 mmHg, P < 0.05), confirming that this is a nonhypertensive model. Treatment with enalapril caused a nonsignificant reduction in MAP in "early"-treated NS rats (110.1 ± 7.7 mmHg, P > 0.05), but produced a significant hypotensive effect when given as a "late" treatment (94.8 ± 9.5 mmHg, P < 0.05) compared with untreated NS rats (124.11 ± 4.9 mmHg). The lack of hypotensive effect of enalapril when administered early to NS rats could possibly be attributed to differences in food and water intake, as well as physical condition between early- and late-treated groups, although these parameters were not determined in the present study. However, we found that urinary volume was comparable in both groups, suggesting that animals given enalapril as an early treatment were not hypovolemic compared with late-treated rats. Administration of eplerenone alone did not affect blood pressure. However, when given in combination with enalapril, it potentiates the hypotensive effects of enalapril (early, from 129.7 ± 4.7 to 99.8 ± 8.3 mmHg, P > 0.05; late, from 137.8 ± 7.3 to 118.4 ± 6.1 mmHg, P < 0.05). It should be emphasized that these results are in line with the blood pressure measurements using the tail-cuff method (data not shown).


Figure 6
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  		Fig. 6. Effects of chronic administration of enalapril and eplerenone alone or in combination (n = 5–7) given as early (top) or late (bottom) therapy on mean arterial blood pressure (MAP) in rats with NS. MAP was measured via cannulation of the carotid artery. *P < 0.05 vs. baseline. #P < 0.05 vs. untreated NS rats (n = 9 and n = 11 in early and late treatment groups, respectively).

 
Renal function. Blood urea nitrogen (BUN) in nephrotic rats was significantly higher at week 6 (41.9 ± 5.5 mg/dl, P < 0.05) compared with normal rats (18.6 ± 0.8 mg/dl). Early or late treatment with enalapril or eplerenone alone did not significantly affect BUN. However, early treatment with a combination of enalapril and eplerenone reduced BUN by 24% (31.6 ± 3.9 mg/dl, P > 0.05).

Plasma lipids. Plasma lipids greatly increased in nephrotic rats at week 6 compared with normal animals (Fig. 7). This was evident by the substantial increase in plasma levels of both cholesterol (from 117.6 ± 36.9 to 652.0 ± 52.1 mg/dl, P < 0.001) and triglycerides (from 120.8 ± 67.6 to 1,067.9 ± 204.6 mg/dl, P < 0.001). Early treatment with enalapril significantly reduced triglyceride levels from 1,067.9 ± 204.6 to 478.9 ± 135 mg/dl, (P < 0.05), but not cholesterol levels.


Figure 7
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  		Fig. 7. Effects of chronic administration of enalapril and eplerenone alone or in combination (n = 5–7) given as early or late therapy on plasma cholesterol levels (top) and plasma triglyceride levels (bottom) in rats with NS. *P < 0.05 vs. baseline. #P < 0.05 vs. untreated NS rats (n = 9 and n = 11 in early and late treatment groups, respectively).

 
Eplerenone alone did not affect plasma lipid levels when given as early treatment and aggravated hyperlipidemia when given as late treatment. Interestingly, combined early treatment with enalapril and eplerenone significantly reduced triglyceride levels from 1,067.9 ± 204.6 to 364.6 ± 114.3 mg/dl (P < 0.05), but reduced plasma cholesterol concentrations only slightly. This trend was also observed when the drugs were given as late treatment.

Plasma electrolytes. The plasma levels of Na+ (PFormula) was slightly lower in NS rats (140.9 ± 1.07) compared with healthy controls (143.6 ± 1.13 mmol/l, P > 0.05). In contrast, plasma K+ (PFormula) was slightly higher in NS rats compared with healthy controls (4.89 ± 0.6 vs. 3.66 ± 0.1 mmol/l, P > 0.05). Enalapril did not affect plasma concentrations of either ion. Eplerenone significantly decreased PFormula to 135.9 ± 1.49 and 134.3 ± 1.4 mmol/l when given either early or late, but had no effect on PFormula. Combination of enalapril and eplerenone lowered PFormula to 138.3 ± 0.74 and 133.8 ± 0.84 mmol/l (P < 0.05), when administered early or late, respectively, but increased PFormula to 7.68 ± 0.41 mmol/l when given as late treatment.

Immunohistochemical analysis. Immunofluorescence studies utilizing an anti-nephrin antibody in normal kidneys showed a finely dotted linear epithelial staining pattern, as has been reported by Holthofer et al. (19) and Yuan et al. (55) (Fig. 8A). In contrast, nephrin staining in the glomeruli of NS rats was attenuated, more dispersed, and clustered. Enalapril prevented the decrease in nephrin staining in NS rats to a large extent (Fig. 8A). Similarly, although to a lesser extent, early treatment with eplerenone prevented the decrease in nephrin immunoreactivity in these animals. Preservation of nephrin expression by eplerenone was more remarkable when given in combination with enalapril (Fig. 8A). In contrast to early treatment, late treatment with either enalapril, eplerenone, or a combination was not significantly effective in preserving nephrin immunoreactivity in NS rats, but produced a slight increase in nephrin abundance compared with untreated NS animals.


Figure 8
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  		Fig. 8. Representative immunofluorescent staining with antibodies against nephrin (A) or podocin (B) in glomeruli from normal rats and animals with untreated NS on day 35 and nephrotic rats treated with enalapril, eplerenone, and enalapril+eplerenone, given as early or late therapy. Normal glomeruli illustrate the normal interrupted linear staining pattern of the slit diaphragms with these antibodies. The staining of untreated NS glomeruli is more dispersed and granular, whereas enalapril alone or in combination with eplerenone given as early but not as late treatment preserved the nephrin and podocin immunoreactivity. Magnification x400.

 
Similar to nephrin, immunostaining with anti-podocin antibodies showed a intense glomerular epithelial staining pattern, as has been reported by Caridi et al. (8) (Fig. 8B). In parallel to nephrin loss, untreated nephrotic rats are characterized by a massive reduction in podocin staining. Early treatment with enalapril, eplerenone, or the combination partially but significantly prevented the disappearance of podocin in nephrotic rats (Fig. 8B). In contrast, late treatment with these drugs either when given alone or in combination, did not substantially improve the podocin immunoreactivity in NS rats. These data clearly indicate that proteinuria is associated with a reduction in nephrin and podocin immunoreactivities in NS and that early treatment with enalapril, eplerenone, or a combination of the drugs reduced proteinuria in correlation with the preservation of these slit diaphragm proteins (Fig. 8, A and B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that pretreatment with enalapril or eplerenone significantly reduces proteinuria in ADR-induced NS in rats. The combination of eplerenone with enalapril resulted in a more significant reduction in proteinuria compared with enalapril or eplerenone alone. Rats with NS exhibited severe disruption of slit diaphragm structure as seen by rapid and profound loss of nephrin and podocin. Treatment with enalapril or eplerenone alone resulted in partial preservation of podocin, but only combined treatment with enalapril and eplerenone was characterized by a dramatic preservation of nephrin and podocin levels, suggesting that concomitant preservation of these molecules is crucial for maintaining the integrity of the glomerular filtration barrier and preventing proteinuria. The beneficial therapeutic effect of enalapril and eplerenone paralleled preservation of nephrin, as determined immunohistochemically, and was sufficient to predict significant antiproteinuric responses. However, since no cause-and-effect relationship was established between the antiproteinuric effects of these drugs and their preservation of nephrin and podocin, it is premature to conclude that ACEI and eplerenone decrease proteinuria via the preservation of these slit diaphragm proteins.

Aminonucleoside-induced nephropathy is widely used as an experimental model of NS with morphological similarities to minimal change and membranous nephropathy (55). The role of slit diaphragm compounds in maintaining permselectivity of the glomerular filtration barrier and preventing injury leading to development of proteinuria in NS indicates that nephrin loss and redistribution are important factors in pathogenesis (9, 16, 19, 31, 55). These data were confirmed in experimental and clinical studies in various glomerular diseases including NS. Inhibition of the RAAS is a well-recognized approach for treating different diabetic and nondiabetic forms of chronic glomerular disease associated with proteinuria (17, 25, 32, 39, 54). Different clinical studies have shown renoprotective and antiproteinuric effects of such treatments. An evaluation of the efficacy of ACEI alone or in combination with the aldosterone blockers in different proteinuric syndromes yields convincing results as well (17, 25, 44).

A previous study by our group (33) has demonstrated that enalapril was superior to losartan in reducing protein excretion in NS rats when given as a pretreatment. In this study, we observed significant differences in antiproteinuric responses in rats treated with enalapril vs. eplerenone or a combination. Absolute as well as cumulative proteinuria measurements were shown to be significantly decreased in rats pretreated with ACEI or eplerenone alone, but even to a larger extent when the drugs were given in combination. Although enalapril and eplerenone produced comparable and significant antiproteinuric effects, only the latter improved plasma total protein and albumin in NS animals. These unexpected differences may suggest, that NS rats treated with enalapril have either less protein production or enhanced protein catabolism.

So far, it is unknown whether the beneficial antiproteinuric effect of eplerenone is primary or secondary. However, our findings, along with those of Nagase et al. (31), suggest that aldosterone, via MR, can cause primary dysfunction of the glomerular filtration barrier in renal diseases characterized by proteinuria. This notion is supported by the observation that aldosterone acts directly on the podocytes and upregulates Sgk1, which most likely mediates glomerular injury and subsequent proteinuria (49).

In the past decade, the role of aldosterone in the pathogenesis of various diseases had received increasing attention. In addition to data obtained in the heart, brain, and blood vessels, evidence has accumulated regarding contribution of aldosterone to kidney damage, especially as a proproteinuric agent (10, 15, 34, 40). Based on different animal studies and preliminary clinical data, aldosterone blockade seems to be a promising approach to reducing proteinuria and delaying progression to chronic kidney disease (13, 14, 36, 44). Although clinical studies performed to date are mostly small, data from open-label trials with a very short follow-up are consistent with a significant antiproteinuric effect of aldosterone blockade, independently of blood pressure changes (46, 47, 56). Antiproteinuric effects of MRB were demonstrated in proteinuric disease states of various etiologies. For instance, Zhou et al. (56) have demonstrated that eplerenone, an aldosterone antagonist, significantly ameliorated proteinuria and nephrosclerosis in an L-NAME/SHR model, independently of hemodynamic effects. Similarly, Schjoedt et al. (47) demonstrated that MRB (spironolactone, at a dose of 25 mg/day) added to conventional antihypertensive treatment in type 1 diabetes mellitus patients, induced a 30% reduction in albuminuria vs. placebo treatment and reduced fractional albumin clearance. Authors of the study conclude that the addition of spironolactone to an antihypertensive prescription reduces blood pressure and may offer additional renoprotection in type 1 diabetic patients characterized by diabetic nephropathy.

Similarly, the role of aldosterone in kidney damage has been studied in animal models of hypertension. Spontaneously hypertensive rats of the stoke-prone substrain (SHRSP) develop severe hypertension and malignant nephrosclerosis (5, 41, 44). ACEI or ARBs were able to prevent development of kidney damage in these animals. When SHRSP animals were treated with spironolactone or placebo for 1 mo, spironolactone-treated animals did not develop proteinuria, which otherwise develops spontaneously in untreated SHRSP group. Therefore, it is hypothesized that some of the positive effects of ACEI are due to inhibition of endogenous aldosterone. In another rat model of hypertension, massive proteinuria and severe hypertensive nephrosclerosis were observed following simultaneous administration of ANG II and an inhibitor of nitric oxide synthase. In this setting, removal of aldosterone by adrenalectomy or aldosterone blockade with eplerenone reduced the severity of proteinuria without altering blood pressure (42, 44).

In support of an important role for nephrin in the pathogenesis of proteinuric states, we found that the severity of proteinuria in NS rats correlated with the abundance of nephrin as visualized by immunohistochemical staining. Loss of nephrin immunostaining that was observed in rats with NS may stem from reduced protein synthesis or exaggerated protein degradation. Our findings concerning reduction of nephrin immunoreactivity in NS rats are in agreement with those of Remuzzi et al. (38), who demonstrated that Heymann nephritis was associated with a marked decrease in nephrin-specific glomerular staining. Similarly, in a model of hypertension and diabetes in the rat, development of albuminuria was shown to be accompanied by reduced gene and protein expression of nephrin (2). In line with our findings in NS, a significant inverse correlation between the degree of albuminuria and expression of nephrin has been found and that specific blockade of the RAAS preserved glomerular nephrin expression in experimental and clinical diabetes mellitus. Untreated diabetic patients show a dramatic reduction in nephrin expression. For example, Benigni et al. (3) have demonstrated substantial downregulation of nephrin and loss of the electron-dense structure of the slit diaphragm. These results overall indicate involvement of nephrin in the pathogenesis of proteinuria in acquired experimental and clinical nephrotic disorders of diabetic and nondiabetic origin.

Another protein important for maintenance and preservation of slit diaphragm structure is podocin. In our study, we have demonstrated that monotherapy with either enalapril or eplerenone partially preserved podocin in rats with ADR-induced NS. We also show that a combination of the drugs produces better preservation of podocin and reduces the proteinuria compared with each drug alone. However, the effect of enalapril in combination with eplerenone on preservation of nephrin was more profound compared with the effect of the combination to preserve podocin. Of note, the antiproteinuric effects were evident only in those cases in which nephrin was also preserved. These findings suggest a role for nephrin in the pathophysiology of proteinuria and provides further insight into its influence on glomerular permselectivity.

In summary, our findings indicate that pretreatment with either enalapril or eplerenone significantly reduces proteinuria in rats with NS; however, a better antiproteinuric effect is achieved by coadministration of eplerenone and enalapril. These antiproteinuric effects may stem from attenuating nephrin and podocin loss, which characterizes the development of nephrotic syndrome in the model of ADR-induced nephropathy.


   ACKNOWLEDGMENTS
 
This work was supported partially by a grant to F. Nakhoul by the Abutbul family in memory of Daniel Abutbul.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Nakhoul, Ambulatory Nephrology Unit, Rambam-Health Care Campus, Faculty of Medicine, Technion, Haifa 31096, Israel (e-mail: f_nakhoul{at}rambam.health.gov.il)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* F. Nakhoul, E. Khankin, and A. Yaccob contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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