An excessive rate of cardiovascular morbidity and mortality and accelerated atherosclerosis characterize chronic renal disease (CRD).1 Similar to cardiovascular disease, the incidence and prevalence of CRD is higher in men than in women.2-4 The rate of progression of CRD of various etiologies is more rapid in men than in women,5-7 and the resistance of kidneys in women to the progression of renal disease is most frequently attributed to estrogens.8,9 Finally in patients with diabetes, it seems that estrogen-related cardiovascular protection is lost, and female patients with diabetes may develop even more severe cardiovascular disease.10,11
Because there is strong evidence that estrogens have cardiovascular and renal protective effects, estrogen therapy should be a rational approach to attenuate cardiovascular disease and chronic renal disease associated with severe forms of the metabolic syndrome. Unfortunately, there are important pharmacologic and clinical factors that may limit the use of estrogens to reduce cardiovascular morbidity and mortality associated with chronic renal disease. These include feminizing effects in men; adverse effects of estrogens on plasma triglycerides especially in patients with nephrosis; and increased risk for development of cancer in target organs, particularly in woman who are premenopausal. Furthermore, recent large prospective clinical studies [Heart and Estrogen/Progestin Replacement Study (HERS) and Women's Health Initiative Study (WHI)] questioned the safety and efficacy of estrogens in primary and secondary prevention of cardiovascular disease.12,13
Evidence suggests that several of the observed cellular effects of estradiol are mediated by its metabolites rather than by estradiol per se.14 In vivo, estradiol is converted to 2-hydroxyestradiol (2-HE), a metabolite with little estrogenic activity, and 2-HE is readily converted (by catechol-O-methyl-transferase) to 2-methoxyestradiol (2-ME), an estradiol metabolite with no estrogenic activity.15 Our previous studies conducted in rodent models of renal disease suggest that the estradiol metabolites of a 2-hydroxylation pathway may be renoprotective.16-19 Of particular importance is the observation that in young, 12-week-old, obese, ZSF1 rats, a model of the metabolic syndrome, 20 week treatment with 2-HE reduces proteinuria and glomerulosclerosis.19 However, in humans diabetic renal disease occurs generally in older patients and is more severe in the elderly. Because our previous studies were conducted in young rats with metabolic syndrome, it is unknown whether estradiol metabolites of the 2-hydroxylation pathway might be renoprotective in the more relevant aged (and more severe) models of renal disease associated with metabolic syndrome. Therefore, an objective of the present study was to determine whether the renal protective effects of estradiol metabolites of the 2-hydroxylation pathway are maintained in older diabetic animals with a severe form of renal disease. To achieve this objective, we examined the effects of 2-ME on the progression of renal disease in 35-week-old, diabetic ZSF1 rats.
Recent data suggest that synthetic analogs of estradiol metabolites may be effective antimitogens and one of the analogs, 2-ethoxyestradiol (2-EE), is reported to be an even more effective antiproliferative agent than 2-ME.20,21 Accordingly, in this study we also examined the renal effects of 2-EE to determine whether it also might afford renal protection.
MATERIAL AND METHODS
A total of 27 male, 35-week-old, obese ZSF1 rats (Charles River Laboratories Inc, Wilmington, MA) was used in this study. Obese ZSF1 rats were generated by Genetic Models Inc (Indianapolis, IN) by crossing lean heterozygous female Zucker diabetic fatty rats (ZDF-/fa) with lean heterozygous male spontaneously hypertensive heart failure rats (SHHF/Mcc-cp, -cp). As we have described recently,22-24 similar to the maternal strain (ZDF diabetic rats), the ZSF1 rats have the metabolic syndrome (ie, hypertension, diabetes, and hyperlipidemia) and develop nephropathy as characterized by massive proteinuria, abnormal renal histopathology (glomerulosclerosis and severe tubulointerstitial and vascular changes), and reduced glomerular filtration rate (GFR). However, ZSF1 rats have more severe hypertension and renal disease and, in contrast to the paternal ZDF diabetic rats, do not develop hydronephrosis20 that may complicate evaluation of renal function and structure.
Animals were housed at 22°C, 45% relative humidity, and 12 hour light/dark cycles; had free access to water; and were fed ad libitum Purina 5008 rodent diet (Pro Lab RHM 3000 rodent diet, PMI Nutrition, Inc, St Louis, MO). Experimental protocols were approved by the University of Pittsburgh Animal Care and Use Committee, and all experiments were conducted in accordance with the University guidelines for animal welfare.
2-ME and 2-EE were purchased from Steraloids, Inc. (Newport, RI), and PEG-400 was purchased from Sigma-Aldrich (St Louis, MO).
Before initiating the treatment, and 3, 6, and 9 weeks into the treatments, animals were placed in metabolic cages and allowed to acclimatize for 2 days before recording body weight and conducting 24 hour measurements of urine volume and food and water intakes. Urine samples were analyzed for proteins (bicinchoninic acid method; Pierce; Rockford, IL) and glucose (Infinity™ Glucose Reagent; Sigma Diagnostics, St Louis, MO), and urinary protein and glucose excretions were calculated. Animals were randomly assigned to be implanted, under halothane anesthesia, with osmotic minipumps (model 2ML4, Alzet, Palo Alto, CA) containing vehicle (polyethylene glycol 400, 2.5 μL per/hour; control group, n = 9), 2-ME (18 μg/kg per hour; 2-ME group, n = 9), or 2-EE (18 μg/kg per hour; 2-EE group, n = 9). Osmotic minipumps were replaced after 32 days.
Six weeks into treatments, animals fasted overnight and then an oral glucose tolerance test (OGTT) was conducted. Blood samples (tail vein) were obtained before and 30, 60, and 120 minutes after an oral glucose load (2 g/kg by gavages), and glucose levels were measured by Precision Q.i.d. Blood Glucose Test kit (Medisense, Inc, Bedford, MA). After 9 weeks of treatment, an insulin tolerance test (ITT) was conducted and total glycosylated hemoglobin levels were measured (A1cNow, Metrika Multi-test-system, STAT Technologies Golden Valley, MN). Each animal was anesthetized with pentobarbital (45 mg/kg ip); short-acting insulin (Actrapid, 0.25 U/kg) was administered intravenously; and blood samples were taken before and 5, 10, 15, 20, 25, and 30 minutes after insulin administration and glucose levels were measured.
Next, animals were instrumented for measurements of renal hemodynamics and excretory function as described previously.23 A short section of PE-240 polyethylene catheter was inserted into the trachea to facilitate breathing. The left carotid artery was cannulated with a PE-50 catheter for blood sample collection and mean arterial blood pressure and heart rate measurements were done via a digital pressure analyzer (Micro-Med. Inc, Louisville, KY). Two PE-50 cannulas were placed in the left jugular vein, 1 for infusion of 14C-inulin (0.035 μCi/20 μL/min), and the other for infusion of saline (50 μL per minute) or anesthetic as needed. Next, the left kidney was exposed, a PE-10 catheter was inserted into the left ureter to facilitate collection of urine, and a flow probe (Model 1RB; Transonic Systems, Inc, Ithaca, NY) was placed on the left renal artery for measurement of renal blood flow. A 45 minute stabilization period was permitted before two 30 minute clearance periods were conducted. Mean arterial blood pressure and renal blood flow were recorded at 1 minute intervals and were averaged during a 30 minute urine collection. A midpoint blood sample (300 μL) was collected to measure radioactivity and hematocrit. Urine volume (UV) was determined gravimetrically, and plasma and urine 14C-inulin radioactivity was measured. Renal clearance of 14C-inulin was calculated as an estimate of GFR. Animals were euthanatized by anesthetic overdose, and testicles were removed and weighed. The right kidneys were rapidly excised and washed in ice-cold phosphate buffer saline and the renal cortex tissue samples were dissected, immediately frozen in liquid nitrogen, and stored for further protein expression analysis.
Kidney Tissue Preparation and Protein Extraction
Frozen tissue samples were ground into a powder with a mortar and pestle. The ground tissues were homogenized in 0.5 milliliters of SDS buffer (50 mM Tris, pH 7.0, 2% SDS, and 10% glycerol) containing protease inhibitors (2 μg/mL antipain, 1 μg/mL aprotinin, 2 μg/mL leupeptin, 1 mg/mL phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 12,000 rpm at 4°C for 10 minutes, and the supernatant was recovered. Protein concentration in the supernatant was measured by the copper bicinchoninic acid method, and samples were stored at −20°C. For renal protein expression analysis, negative controls (ie, lean, male ZSF1 littermates, n = 7) were also included.
Proteins were solubilized at 60°C for 15 minutes in Laemmli sample buffer. Samples (30 μg protein/well) were loaded onto a 7.5-10% acrylamide gel and subjected to SDS-PAGE using the Bio-Rad minigel system. Proteins were then electroblotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membrane was blocked with 5% milk for 1 hour and incubated for 3 hours at room temperature or at 4°C overnight with the first antibody: Proliferating cell nuclear antigen (PCNA) rabbit polyclonal antibody (catalog # sc-7907), vascular endothelial growth factor (VEGF) mouse monoclonal antibody (catalog # sc-7269), transcription factor nuclear factor kappa B subunit 50 (NFκB-50) mouse monoclonal antibody (catalog # sc-8414), and NFκB subunit 65 (NFκB-65) mouse monoclonal antibody (catalog # sc-8008) were obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Membranes were probed with β-actin (Sigma-Aldrich, St. Louis, MO) for 1 hour to determine loading efficiency. Membranes were washed 3 times in phosphate buffer saline containing 0.5% Tween 20 solution and then incubated at room temperature for 1 hour with horseradish peroxidase-conjugated donkey antiimmunoglobulin G secondary antibody (Amersham, Arlington Heights, IL) at 1:5000 dilution. Densitometric analysis was performed by using ImageQuant TL (Amersham Biosciences, Inc, Piscataway, NJ) and band densities were normalized to β-actin.
All data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using the Number Cruncher Statistical software program (Kaysville, UT). Group comparisons for data from metabolic studies (repeated measurements) were performed using a 2 (2F) hierarchical analysis of variance (ANOVA), followed by a Fisher's LSD test for post-hoc comparisons. Comparison of data from acute experiments and from baseline metabolic cage experiments (single point data) was performed by 1-factor ANOVA (1F-ANOVA). The probability value of P < 0.05 was considered statistically significant.
As shown in Table 1, at baseline (35 weeks of age and before initiation of treatments) control and 2-ME and 2-EE groups had similar body weights, food and water intakes, urine volumes, and urinary excretion rates of glucose and protein. This table also shows that 35-week-old ZSF1 rats have polydipsia and polyuria and spill large quantities of protein and glucose into their urine. The overt proteinuria (>1 g/day; Figure 3, Week 0) significantly increased over the course of the study. As renal disease progressed and general health declined, there was significant (∼40%) reduction in food consumption in the control group. This may explain the time-dependent reduction in glycosuria in control animals (Table 1). Therefore, the effects of treatments on urinary glucose excretion are also presented as percent change from the baseline (Figure 2A).
Nine-week treatment with 2-ME or 2-EE had no effects on body weight (Figure 1). 2-ME and 2-EE reduced urine volume, fluid intake (Table 1), and urinary glucose excretion (Table 1, and Figure 2A). It is important to note that 2-EE tended and 2-ME significantly reduced glycosylated hemoglobin (Figure 2C). Also, 2-ME, but not 2-EE, reduced fasting glucose levels (235 ± 13, 203 ± 15 and 248 ± 16 mg/dL, Cont, 2-ME and 2-EE groups, respectively). 2-Ethoxyestradiol had no effect on OGTT and ITT, whereas 2-ME tended to improve OGTT and ITT (Figure 2 B and 2C).
Treatment with 2-ME and 2-EE had renoprotective effects, with 2-EE being somewhat more effective in animals with renal disease associated with metabolic syndrome. In overtly proteinuric animals, 2-ME prevented further increase in proteinuria, whereas 2-EE in a time-dependent manner reduced proteinuria (Figure 3). Both 2-ME and 2-EE reduced renal hypertrophy, attenuated the reduction in renal blood flow and GFR, and decreased renal vascular resistance (Table 2). 2-ME and 2-EE had no effect on heart rate and blood pressure. The renoprotective effects occurred in the absence of feminizing effects (ie, 2-ME and 2-EE had no effects on testicle weight; data not shown).
VEGF functions to induce angiogenesis and to increase microvascular permeability. The major sources of VEGF in the kidney are tubular epithelia cells and glomerular podocytes, which produce VEGF in a constitutive manner. In the adult kidney VEGF lacks its angiogenic properties, but it retains its vascular permeability effects, which have been linked to proteinuria in diabetic nephropathy. It is important to note that in a diabetic environment there is increased renal expression of VEGF. As shown in Figure 4 (left bar graph) densitometric analysis revealed significantly increased protein expression of VEGF in renal cortical tissue of obese control animals compared with their lean littermates. Both 2-ME and 2-HE significantly (P < 0.05) reduced VEGF protein expression.
PCNA is a key protein controlling the growth state of cells,25 and high PCNA levels indicate DNA replication. Compared with their lean littermates, obese diabetic animals had increased proliferative response (Figure 4, right bar graph), and treatment with 2-ME and 2-EE significantly decreased PCNA protein expression in the renal cortex of diabetic animals.
NFκB is a family of transcription factors that are usually present as dimers, the most common being the p50/p65 heterodimer. By influencing the release of more than 40 inflammatory cytokines and oncogenes, NFκB transcription factors are involved in the control of a large number of cellular and organism functions, including inflammation, growth, and apoptosis. Obese, diabetic ZSF1 rats have increased expression of NFκB subunits p50 and p65 (Figure 5) compared with their lean littermates. This is in accordance with an increased level of NFκB protein in diabetic rats and patients with diabetes.26 2-ME and 2-EE significantly (P < 0.05) decreased the expression of NFκB-50 and NFκB-65 (Figure 5).
The main finding of this study is that 2-ME and the synthetic analog 2-EE provide renoprotection in aged, obese, diabetic rats with severe nephropathy. This implies that estradiol metabolites of the 2-hydroxylation pathway and their synthetic analogs may be useful for preventing renal disease in older patients with more severe diabetic renal disease.
The present study is in accordance with our recent studies in rodent models of renal disease. In this regard, we find that in the chronic puromycin aminonucleoside (PAN)-induced renal disease model, treatment with 2-HE retards the progression of renal disease.16 Chronic treatment with 2-HE significantly attenuates PAN-induced decrease in glomerular filtration, reduces proteinuria and the elevated blood pressure, and inhibits glomerular remodeling and sclerosis. Also, 2-ME and its metabolic precursor 2-HE exhibit renal protective effects in a rat model of chronic nitric oxide synthase (NOS) inhibition-induced renal injury.17 For example, prolonged NOS inhibition with N-ω-nitro-L arginine induces severe proteinuria, markedly reduces GFR, causes proliferative and inflammatory responses in the kidney, and is associated with a high (75%) mortality. 2-ME, but not 2-HE, significantly attenuates the reduction in GFR in the NOS inhibition model. Whereas 2-HE only delays the onset of proteinuria, 2-ME prevents NOS inhibition-induced proteinuria, and both metabolites reduce renal inflammatory and proliferative responses and markedly decrease mortality. Finally, both 2-ME and 2-HE inhibit proteinuria induced by chronic infusion of angiotensin II.18
We propose, and the present study and previous data suggest, that nonestrogenic metabolites of estradiol, or their synthetic analogs, may provide more benefit in the metabolic syndrome than traditional estrogens. The present study demonstrates that both 2-ME and 2-EE evoke considerable renoprotective effects and exhibit strong antiinflammatory and antiproliferative effects in the kidney in aged animals that already express severe renal disease. These protective effects are entirely consistent with previous studies of the effects of 2-HE in rodent models of renal disease.16-19 How do 2-ME and 2-EE afford renoprotection in severe forms of the metabolic syndrome?
It is conceivable that improved insulin sensitivity and glucose homeostasis would attenuate diabetic renal injury. Previously we have shown that in the same model of diabetic renal disease, 2-HE significantly improves glucose control in young, obese ZSF1 rats (ie, reduces both plasma glucose levels and glycosylated hemoglobin). It is well established that the peroxisome proliferator-activated receptor gamma (PPARγ) agonists improve insulin sensitivity.27 It is interesting that 2-ME has structural similarities with PPARγ ligands, and a preliminary report suggests that 2-ME may interact with the PPARγ system.28 Therefore, in the present study we examined the effects of 2-ME and 2-EE on glucose homeostasis. It is surprising that 2-ME and 2-EE had variable effects on glucose homeostasis. Both 2-ME and 2-EE reduced glycosylated hemoglobin (HbA1c) and glycosuria, and 2-ME, but not 2-EE, reduced fasting glucose levels and tended to improve insulin tolerance test results. Nonetheless, the impact of these effects on renal function is questionable. First, 2-ME and 2-EE-treated animals still had significantly elevated HbA1c and fasting glucose levels. Second, the effect of 2-ME on fasting glucose and ITT were at best mild. Finally, 2-EE had no effects on fasting glucose and ITT, yet it was somewhat more renoprotective than 2-ME in aged ZSF1 rats. Therefore, most likely renoprotective effects of 2-ME and 2-EE were mediated largely via mechanisms unrelated to changes in diabetic status.
Diabetic animals and patients have increased expression of the transcription factor NFκB,24 and NFκB apparently contributes to the development of renal diseases including diabetic nephropathy.29 For example, previous studies report that inhibition of NFκB reduces renal injury in rodent models of proteinuric nephropathy.30,31 The present study shows that obese, diabetic animals have increased NFκB expression and that both 2-ME and 2-EE reduce NFκB expression. These findings suggest that the renal protective effects of 2-ME and 2-EE are related to the ability of these compounds to reduce expression of NFκB.
The renoprotective effects of 2-ME and 2-EE in the metabolic syndrome in aged animals may be mediated in part via changes in VEGF expression. Our immunoblotting analysis reveals increased expression of VEGF protein in the renal cortex of obese ZSF1 animals compared with their lean littermates, and 9-week treatment with 2-ME or 2-EE reduces the renal cortex expression of VEGF. The increased expression of VEGF in obese ZSF1 rats is not surprising because the diabetic milieu contains factors (such as high glucose and advanced glycosylation end products, high lipids, and reactive oxygen species) that may activate VEGF expression.32 Moreover, our results in this regard are consistent with reports of increased VEGF gene expression in streptozotocin and Zucker diabetic fatty rats.33-35 Currently, there is compelling in vivo evidence for the role of VEGF in development of diabetic renal disease in animal models and patients with diabetes.32,36 Treatment with anti-VEGF antibodies improves early renal dysfunction in diabetic rats by reducing hyperfiltration and albuminuria; attenuates the reduction in creatinine clearance; and ameliorates long-term renal changes in kidney weight, glomerular volume, basement membrane thickness, and total mesangial volume.37,38 In contrast to its protection of the integrity and repair of endothelium, recent reports suggest that VEGF enhances arteriosclerosis in hypercholesterolemic mice and in a rat model of chronic NOS inhibition-induced renal disease.39,40 Furthermore, in vivo angiotensin II-induced renal damage involves increased VEGF expression and related inflammation.41,42 It is worth mentioning that in the chronic NOS inhibition model15 and in the angiotensin II-induced renal damage model,16 as well as in the present study in hyperlipemic ZDF1 rats, 2-ME exhibits a strong antiproteinuric effect and attenuates cardiovascular and renal injury.
The reduction of renal expression of VEGF in this study by 2-ME and 2-EE is not surprising because 2-ME is known to exert antiangiogenic properties and to inhibit the expression of VEGF in tumor cell lines and in vivo,43,44 although at much higher dose levels than those used in this study. Activated glomerular mesangial cells and macrophages are the main sources of VEGF production in the diabetic kidney. Notably, in vitro 2-ME and its metabolic precursor 2-HE inhibit mesangial cell proliferation,12,14 and in vivo both metabolites abolish interstitial and glomerular macrophage influx (ED1+ cells) and inhibit glomerular and tubular cell proliferation and collagen IV synthesis.14,15 Therefore, the antiinflammatory and antiproliferative effects of 2-ME and 2-EE most likely account for the reduction in VEGF expression.
In summary, experimental and clinical data suggest that estrogens have cardioprotective and renoprotective effects. However, there are important pharmacologic and clinical factors that may limit the use of estrogens in the prevention or treatment of cardiovascular disease and chronic renal disease in patients with the metabolic syndrome. This study provides unequivocal evidence that 2-ME, a nonestrogenic metabolite of estradiol, and the synthetic analog 2-EE have renoprotective effects in renal disease associated with the metabolic syndrome. It is remarkable that the beneficial effects (ie, reduced proteinuria and attenuation of time-related decline in renal function) are produced by short-term (9-week) treatment in aged, 35-week-old diabetic animals that at the beginning of treatment expressed severe and progressive renal failure. A long-acting formulation of 2-ME is now in Phase I clinical trials for pulmonary hypertension. The present data suggest that this formulation of 2-ME might provide renoprotection in patients. This study also warrants further investigation of analogs of estradiol metabolites for treatment of chronic renal disease.
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