The effects of supraphysiological levels of cortisol alone or in combination with physiological levels of selenomethionine on kidney morphology.

Chronic increased levels of glucocorticoids (GC) can result in hypertension, which may ultimately lead to impairment of renal function. Recent studies have shown GC excess can elicit serious adverse effects on the vascular system. Glucocorticoid excess causes over production of reactive oxygen species (ROS) and disrupts nitric oxide availability in the vascular endothelium, leading to vascular complications in patients with GC excess. Increases in reactive oxygen species are implicated in a number of disease processes and strategies that are directed at counter balancing the oxidative process could have a potential role in clinical medicine. The use of antioxidants has been shown to reduce ROS as well as renal tubular injury in cases of increased glucocorticoid administration in vitro. The objectives of this study were: (1) to establish an animal model of increased glucocorticoid levels by sustained delivery and (2) to determine if sustained delivery of selenomethionine in combination with glucocorticiods can protect kidney tubular structures. Sixteen female rats were divided into four equal groups (control and 3 experimental groups implanted with tricalcium phosphate lysine drug delivery systems (TCPL) charged with either 50mg selenomethionine (Se), 50 mg cortisol (C), or 50 mg of both C and Se). At the end of 24 days, the rats were sacrificed and both kidneys were removed for histological analysis. Quantitative analysis was performed on serum calcium levels, body weights and kidneys weights in all groups. Kidney slides were evaluated for changes in kidney morphology namely change in area and width. Sustained release of Se and Se + C resulted in a significant reduction of glomerular area (p < 0.05). Data obtained indicated that C, Se and Se + C administration caused a reduction in serum calcium levels compared with control. The reduction may be in part to changes in calcium-filtered load, changes in glomerular filtration rates or interference of calcium absorption from the gut. In conclusion, high levels of cortisol will modify kidney structure and possibly alter blood pressure.

Cortisol is the major adrenal glucocorticoid (GC) in humans and most mammals, and the kidney is the main site for cortisol metabolism. Rodents do not express adrenal 17 hydroxylase, which is responsible for the production of cortisol, and therefore, corticosterone is the major GC in rats. The enzyme 11[beta]-hydroxysteroid dehydrogenase type 2 (11[beta]-HSD2) converts corticosterone to 11-dehydrocorticosterone in the distal convoluted tubules and cortical collecting ducts in rats, and cortisol to cortisone in humans. The 11[beta]-HSD2 is thought to prevent endogenous glucocorticoids from exerting mineralcorticoid-like effects (Bailey, Unwin, and Shirley, 2001). Impairment of the enzyme has led to sodium retention, volume expansion and hypertension (Stewart et al., 1988) and is believed to contribute to the pathophysiology of hypertension. Defective renal cortisol metabolism could contribute to some of the consequences of renal disease (hypertension and sodium retention) or modify other (hyperkalaemia and decreased glomerular filtration rate (GFR)) (Mangos et al., 2003). In conditions such as Cushing's disease or ectopic adrenocorticotrophin production where GC levels in the plasma are high, the 11[beta]-HSD2 is thought have reached saturation kinetics as opposed to impairment. Cortisol is responsible for increasing renal vascular resistance (RVR), GFR and filtered fraction (FF) while lowering renal blood flow. In addition to these hemodynamic properties, glucocorticoids have additional actions on sodium/phosphate cotransport, sulphate cotransport and amino acid transport. Patients with Cushing's syndrome (CS) have been reported to have a high incidence of kidney stone formation (nephrolithiasis) (Ross et al., 1966). Changes in renal clearance for calcium may also reflect the increase in renal stone formation in CS patients. The underlying pathogenesis in the development of nephrolithiasis in Cushing's syndrome is poorly understood. However, it is thought to occur via a glucocorticoid-dependent mechanism.

Recently, in CS it has been shown that cortisol can stimulate sodium and water reabsorption and increase blood pressure. The dogma is that GC will increase blood pressure by acting on renal type 1 mineralcorticoid receptors to produce water and salt retention. However, experiments by Montrella-Waybill et al. (1991), showed that spironolactone, a competitive antagonist of mineralcorticoids, did not affect cortisol induced sodium retention or blood pressure elevation suggesting that cortisol may not necessarily innervate mineralcorticoid receptors. The data suggest that excess cortisol increases sodium retention via a different mechanism. Faggiano et al. (2002) demonstrated that CS patients have significant changes in serum and urinary concentration of several amino acids and changes in renal clearance of some specific amino acids. Their study also showed that normalization of cortisol levels restored the amino acid profile. Patients with high serum levels of cortisol experience a vast array of complications including decrease healing, osteoporosis, hyperlipidemia, kidney stones and diabetes to name a few.

Ogasawara et al. (1999) studied systemic and nonsystemic stress responses in the male rat kidney. They showed acutely stressed rats had increased serum corticosterone and renal cortical reduced glutathione (GSH) levels. They concluded from their study that increases in endogenous GC directly stimulate the defense mechanism within the kidney by increasing GSH. Glutathione peroxidase is an important enzyme in cellular antioxidant defense systems, detoxifying peroxides and hydroperoxides. As a component of the GSH cycle, it protects from reactive oxygen metabolites. Selenocysteine is present at the catalytic site of glutathione peroxidase, and selenium availability regulates glutathione peroxidase enzyme activity and ultimately GSH. The finding by Ogasawara et al. (1999) are important and suggests the possible need for nutritional supplementation of selenomethionine in chronically stressed patients such as those with CS. Nutritional supplementation may reduce the risk of many important complications associated with increased levels of cortisol. For example, supplementation with selenium, vitamin C, zinc, and fluoride might help offset the inhibitory effect of cortisol on fibroblast and osteoblast function. Antioxidant nutrients could support humoral immunity and neutrophil function. Supplementary selenium could reduce the risk of nephrocalcinosis and nephrolithiasis associated with increased cortisol as seen in CS. Therefore, the objectives of this research was to determine the effect of sustained increased levels of cortisol alone and in combination with selenium for 24 days in adult male rats. Specifically, to determine the effects of these compounds on body weight, serum calcium levels as well as kidney morphology.

MATERIALS AND METHODS

Experimental Design. A total of 16 male adult Sprague Dawley female rats from Harlan Suppliers were randomly divided into four equal groups. A control group receiving no treatment and 3 experimental groups administered sustained delivery devices were used. Animals in groups II, III, and IV were surgically implanted with a TCPL drug delivery devices containing either 50 mg selenomethionine (Se), 50 mg of cortisol (C) or 50 mg of both cortisol and selenomethionine (Se + C). During the investigation, blood samples were obtained via the tail artery bi-weekly and X-rays were performed to evaluate the resorptive properties of the drug delivery system.

Ceramic Capsule Design. Tricalcium phosphate was prepared by the method established by Bajpai and Benghuzzi (1988). The TCP material was sieved through a series of Tyler sieves to obtain sizes of TCP ranging from 1-38 [micro]m. The TCP material was processed by first mixing 70% (w/v) calcium nitrate with 100 mL of one molar ammonium hydroxide. This solution was labeled solution 1. A second solution was prepared separately by adding 27% ammonium hydroxide and 16% ammonium hydrogen phosphate. This solution was labeled solution 2. Solution 1 was slowly added to solution 2 over a period of 20 minutes using a funnel. A precipitate formed and was harvested and centrifuged at 2000 rpm for five minutes, washed three times in water, and then resuspended in 3% ammonium sulfate. The resultant material was then dried for approximately four hours and then calcined for one hour at 1150 [degrees]C. The calcined material was placed on the Tyler sieve to obtain TCP particles ranging from 1-38 [micro]m. The TCP powder (1 gram) was combined with 30 mg of lysine for strength and either 50 mg of C, 50 mg of Se, or 50 mg Se + 50 mg of C and pressed with a compression load of 2,500 kgs.

Surgical Procedure. Implantation of ceramic delivery systems was accomplished according to surgical guidelines approved by the University of Mississippi Medical Center IACUC committee. To ensure sterility of capsules and equipment, they were processed in ethylene oxide overnight. The abdomens of each rat were shaved and scrubbed with povidone iodine. Animals were administered intraperitoneally (IP) a mixture of Ketamine/Xylazine for anesthesia. Using standard aseptic techniques the capsules were implanted intraperitoneally through a 1.5-cm incision. The peritoneum was then closed using a 3-0 silk suture, and the skin utilized four to six wound clips (9mm). Postoperatively animals were injected with 0.1 cc of 300,000 units of Penicillin-G/Procaine (IM). Wounds were cleansed daily with povidone iodine. Wound clips were removed after ten days using a surgical staple remover.

Histopathological Evaluation. After 24 days, the animals were euthanized and the organs were removed. The kidneys, adrenals heart, liver, spleen, liver and lung samples were removed and the organs wet weights were obtained and recorded. Organs were placed in 10% buffered formalin. The organs were processed through a dehydration method and then embedded in paraffin blocks. The blocks were then sectioned. Sectioning was performed using a Leica microtome to produce 5 [micro]m sections. The sections were floated in a water bath and allowed to adhere to 3 X 3 glass slides and allowed to dry. Slides were then stained using standard hematoxylin and eosin staining procedure for further analysis.

Histomorphometric Analysis. Utilization of Image Pro Software was the primary means of obtaining morphometric analysis of glomerular changes. After proper calibration of the software, images at 40X and 10X were obtained. A total of 4 tissue sections per slide were examined for differences in area and width. Five measurements from each glomerulus were made utilizing the Image Pro Software. A total of 160 measurements were obtained and recorded per group.

Serum and X-ray Analysis. Blood samples were obtained semi-weekly and X-rays were performed to evaluate the efficiency of the drug delivery system. During the investigation, approximately 500 [micro]L of blood was collected into a 3.5 mL autosep blood collection tube and kept on ice for 1 hour. The tubes were then centrifuged at 2,500 rpms for 30 minutes. Serum was collected and placed in labeled microcentrifuge tubes and stored at -20 [degrees]C until further analysis.

Calcium Analysis. Calcium serum levels were determined using an endpoint determination assay designed by Pointe- Scientific pre and post operatively. Five milliliter glass culture tubes were labeled "reagent blank," "standard calcium (10 mg/dL)," "control," and "sample." One milliliter of reagent was placed into each tube. Ten microliters (10 [micro]L) of serum were added to the respective tubes. The solution was then mixed and allowed to incubate at room temperature for at least one minute. The spectrometer was blanked with the reagent at 650nm. Then the absorbencies of the samples and supernatant were read and recorded and the results were calculated (Pointe Scientific 2001). Total serum calcium levels were calculated based upon the following equation:

Absorbance of sample/Absorbance of standard X [calcium standard] = calcium (mg/dL)

Data Analysis. Statistical data was analyzed using Jandel's Sigma Stat Software and Microsoft Excel. Slidewrite Software was utilized to convert results to graphs. Descriptive statistics were used to determine the mean, standard deviation, standard error of the mean, range, median, mode and quartile data. Analysis of variance was used initially the compare the four groups to determine differences between the groups. Parametric ad hoc tests were used to determine significant changes between groups. Significance are reported at 95% confidence.

RESULTS AND DISCUSSION

Body Weights. Results from this study indicated that sustained delivery of cortisol and selenomethionine for 24 days did not provoke changes in body weight (Figure 1). Se treated animals' shows a slight insignificant increase in the final body weight analysis (Figure 1B). Control and cortisol body weights were consistent and did not change throughout the experiment. Cortisol excess in man is characterized by abdominal obesity, hypertension, glucose intolerance, and hyperlipidemia as well as contributes to an increase risk for cardiovascular disease (CVD) (Andrews and Walkers, 1999; Colao et al., 1999; Lindholm et al., 2001; Boscaro et al., 2001). In rats, GCs seem to be important in the development of obesity, which can be attenuated with GC receptor antagonists. Livingstone et al. (2000) showed stressed Zucker rats were heavier, hypercorticosteronemic and excreted more corticosterone metabolites than non-stressed or lean rats. The data from our experiment show that sustained continuous administration of cortisol (2-5 ng/day) was not able to increase body weights above the control group for the duration of the experiment. However, it was noted during necropsy a must greater increase in omental fat in the both the C and C + SE treated groups when compared with control and SE treated animals. The data suggests the possibility that GCs cause redistribution of fat similar as to that which occur in CS. In CS patients there is a gain in fat in the back of the neck, supraclavicular area and face and loss of fat from the extremities. Czech and Fain (1972) proposed that the adipose tissue hypertrophies in CS and responds preferentially to the lipogenic and antilipolytic actions of the elevated concentrations of insulin evoked by GC induced hyperglycemia. They also suggest that the adipocytes in the extremities are less sensitive to insulin and more sensitive to the lipolytic effect of other hormones.

[FIGURE 1 OMITTED]

Serum Calcium. Sustained delivery of C, Se and Se + C by TCPL significantly (p < 0.05) reduced the serum calcium levels when compared with control animals. Se and combination of Se and C caused the greatest depression of serum calcium after 1 week post-op. Serum calcium levels four weeks post-op in selenium treated animals showed the greatest increase back toward control calcium levels (Figure 2). The mechanism of increase is probably due to the body attempting to restore calcium balance by either resorption of calcium via the bone or by altering calcium excretion. It is thought during GC excess abnormal handling of cations, sodium (Na(+)) and calcium (Ca(2+)), occur either primarily or in part by the kidney tubule (Ferrari, 2003). Our data does not show changes in serum calcium concentration in C treated animals from the control suggesting that the sustained release of C was not at a high enough concentration to saturate the enzymes in the kidney which protect against GCs having mineralcorticoid activities. In humans, the mineralcorticoid receptor (MR) in the kidney is protected from GC occupation by the enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 betaHSD2), which converts cortisol to a receptor-inactive form, cortisone. This enzyme allows lower levels of aldosterone to be the agonist of the MR in spite of higher circulating levels of cortisol. Kinetic properties of 11 betaHSD2 suggest saturability of this enzyme is achieved with high plasma cortisol levels which can result in overstimulation of the MR by cortisol by GC excess.

The significant decrease in serum calcium concentrations in Se and Se + C treated animals was unexpected. Little information on serum calcium levels are available in the literature to date. However, there is significant research showing a protective effect of selenium for cisplastin induced nephrotoxicity. Francescato et al. (2001) showed lipid peroxidation is one of the mechanisms by which cisplatin induces oxidative injury to kidney tissue. They demonstrated cisplastin increased renal malondialdehyde, renal glutathione, and serum creatinine and decreased creatinine clearance. Selenium treatment decreased the effect of cisplastin on serum creatinine, and renal malondialdehyde levels, but selenium did not affect the other parameters with the exception of kidney necrosis. The concentration of selenium administered was 2 mg/kg (equivalent to 500 [micro]g in a 250 g rat) body weight by gavage which was 1000-fold higher than our daily administered dose of 5 ng/day. However, administration by gavage undergoes first pass metabolism whereas sustained delivery via TCPL avoids first pass metabolism. The bioavailability of selenium may be greater via sustained release and account for the differences observed.

Kidney Histopathology. Average glomerular diameter was determined by taking the average five reference points measured from one point of the glomeruli across the glomeruli to a second point (Figure 3A). A total of eighty measurements were taken per group at 40X magnification. The results show a significant change in glomerular diameter in the Se and the Se + C treated animals when compared to control and C treated animals. The differences were significant at p < 0.001. Histological examination showed a reduction in the area of glomeruli as a result of an increased thickening of the inner epithelial layer. Calculation of the area showed significant reduction in the area of Se and Se + C treated animals (Figure 3B). These changes may be due to a compensation mechanism to reduce the renal clearance of calcium by decreasing the glomerular filtration rates in order to reestablish serum calcium balance. Histomorphometric analysis of the tissue revealed changes in glomerular area in Se an Se + C treated animals that may alter GFR. This data is consistent with data reported in the literature for hyperoxaluria rat model. When given selenium and vitamin E to protect against renal injury due to oxidation, urinary excretion of oxalate and calcium were normalized (Santhosh et al., 2003). Figure 4 shows representative photographs of the kidney tissue from the various treated groups showing changes in the area. A more in depth screening of the kidney tissue needs to be performed. Figure 5 shows an increased magnification of the kidney representing proximal/distal tubules, juxtaglomeular apparatus, and macula densa.

[FIGURE 2 OMITTED]

CONCLUSION

The results indicate TCPL delivery systems are capable of administering corticosteriods alone or in combination with Se for 24 days. Results also suggest that sustained release of C and Se via TCPL system resulted in a significant change in glomerular area when comparing all groups. The results also conclude both Se and Se + C administration affected calcium levels. The body's homeostasis mechanisms for maintaining calcium concentration were upregulated in Se treated animals. This compensation may be attributed to alterations in GFR.

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Michelle Tucci, Ham Benghuzzi, James Hughes, Russell Lyon, and Stevie Adams

University of Mississippi Medical Center Jackson, MS 39216