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1Department of Medicine, Division of Endocrinology and Metabolism, Georgetown University, Washington, District of Columbia 20057; and 2Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
Submitted 24 February 2004 ; accepted in final form 4 August 2004
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ABSTRACT |
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275 g) were fed one of four diets for 4 wk (n = 6/group): 1) control, 2) 65% fructose, 3) control + added NaCl (2.59%), or 4) fructose + NaCl. In study 2, 5% butter (fat) was added to the above four diets. Both fat and NaCl, but not fructose, caused modest rises in MAP (5–10 mmHg) and increased the day-to-night ratio in diastolic blood pressure. NaCl or fructose increased kidney size. Creatinine clearance was increased by salt or fat, and fractional excretion of sodium was decreased by fat. In study 1, high NaCl markedly reduced plasma renin and aldosterone and its regulated proteins in whole kidney, i.e., the thiazide-sensitive Na-Cl cotransporter and the 
- and 
(70-kDa band)-subunits of the epithelial sodium channel. These effects were blunted by fat. Fructose increased the abundance of the sodium phosphate cotransporter, whereas it decreased the bumetanide-sensitive Na-K-2Cl cotransporter and aquaporin-2. Overall, doubling of dietary fat appeared to impair dietary sodium adaptation, i.e., blunt the downregulation of aldosterone-mediated effects, thus allowing blood pressure to rise at an accelerated rate. insulin resistance; natriuresis; diuresis; NaPi-2; sodium hydrogen exchanger 3; BSC1; TSC; epithelial sodium channel
We used parallel semiquantitative immunoblotting to assess the effects of various diets on eight major renal sodium transport proteins: the -1 subunit of Na-K-ATPase (48), the sodium-phosphate cotransporter (NaPi-2) (8), the sodium hydrogen exchanger (NHE3) (3), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2 or BSC1) (28), the thiazide-sensitive Na-Cl cotransporter (NCC or TSC) (28), and the -, 
-, and -subunits of the epithelial sodium channel (ENaC) (23). In addition, because our recent studies (14) demonstrated an increase in blood pressure in our rat model of hyponatremia due to water retention, we also examined the regulation of three major renal water channel proteins [aquaporins (AQPs) 1–3] (33, 42) in these rats. We hypothesized that increased protein expression or cellular abundance of any of these proteins might contribute to inappropriate sodium and water retention.
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METHODS |
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275 g, Taconic Farms, Germantown, MD) were randomly assigned to one of four different dietary treatments (n = 6/group). Rats were fed custom-formulated diets for 28 days (Harlan-Teklad, Madison, WI). In study 1, the four diets were: 1) control [40% cornstarch (by weight), 15% maltodextrin, 10% fructose]; 2) fructose (65% fructose); 3) diet 1 with 2.59% NaCl; and 4) diet 2 with 2.59% NaCl. For study 2, the same four base diets were fed with the addition of 5% unsalted butter (by weight) to all four diets. We fed a level of fructose the same or similar to what many other researchers (22, 24, 50) had shown raised mean arterial pressure (MAP) or systolic blood pressure by 10–30 mmHg. For the high-NaCl diets (diets 3 and 4), we chose to feed 10x the level in diets 1 and 2, 0.259%, so the diets remained equally palatable and growth would likely not be affected. In study 2, we chose to approximately double the amount of calories derived from fat (from 12 to 21%) with this diet. Base diets were ground, and butter (study 2 only), 61% water, and 1% melted agar (Sigma, St. Louis, MO) were added to make a gelled form of the diets that was easier to feed, as previously described (17). Five to seven days before initiation of the dietary treatments, a subset of rats (5/treatment group) was implanted with radiotelemetry blood pressure transmitters (Data Sciences International, St. Paul, MN). Briefly, under isoflurane anesthesia (IsoFlo, Abbot Laboratories, North Chicago, IL), the tip of the fluid-filled catheter of the radiotransmitter, which detects the pressure, was advanced into the aorta via an incision in the femoral artery. The body of the radiotransmitter was secured in a pouch under the skin near the left hindlimb. Blood pressure was measured for 10 s at 10-min intervals for the entire study. All animals were maintained at all times under conditions and protocols approved by the Georgetown University Animal Care and Use Committee, an American Association for Accreditation of Laboratory Animal Care-approved facility.
In the last week, urine was collected for 3 days by housing rats individually in Nalgene metabolic cages (Harvard Apparatus, Holliston, MA). At this time, daily feed and water intake records were made. Rats were euthanized by decapitation, and trunk blood was collected into both heparinized and K3-EDTA tubes (Vacutainer, Becton-Dickinson, Franklin Lakes, NJ). A measurement of blood glucose was obtained by glucometer (TheraSense, Freestyle, Alameda, CA) at the time of death on trunk blood. Both kidneys were rapidly removed and either frozen on dry ice for later processing or immediately homogenized.
Tissue and urine analyses.
Urine and plasma were analyzed for sodium, creatinine, aldosterone, insulin, and vasopressin, as previously described (9). Renin activity was determined by a chemical conversion of angiotensinogen to angiotensin I, which was detected by radioimmunoassay (Diagnostic Products, Los Angeles, CA). True plasma triglycerides were determined by colorimetric analysis (Sigma kit TR0100). The left kidney from each rat was homogenized whole, and aliquots were prepared for immunoblotting, as previously described (16, 17). Separate immunoblots were done for all eight sodium transport proteins and all three AQPs for both studies. Our detailed semiquantitative immunoblotting protocol and the production, affinity purification, and characterization of the primary antibodies against NHE3, NaPi-2, NKCC2, NCC, -, -, and -ENaC, as well as, AQPs 1–3, have been previously described (18, 19, 21, 30–32, 37, 41). The mouse monoclonal antibody to the -1 subunit of Na-K-ATPase was obtained from Upstate Biotechnology (Lake Placid, NY).
Statistics. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test or Kruskal-Wallis ANOVA on ranks followed by Dunn’s multiple comparisons test (when data were not normally distributed or variance was different between groups). Multiple comparisons tests were only applied when a significant difference was determined in the ANOVA analysis, P < 0.05. For tables, the superscripts A, B, and C were assigned to the means based on the outcomes of the multiple comparisons test, so that means with letters in common were not significantly different from each other. The letter "A" was applied to the mean with the highest nominal value and any other means with an "A" superscript would be not different from it. "B" or "C" was applied to progressively lesser means, so that means assigned "A" were significantly greater than means assigned "B" but means assigned "AB" would not be different from means assigned either "A" or "B." Three-way ANOVA [carbohydrate (CHO) type x NaCl level x fat] or two-way ANOVA (CHO type x NaCl level) was also used to determine the significance of these factors, as a whole, on the parameters being measured (Sigma Stat, Chicago, IL).
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RESULTS |
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To evaluate potential differences in diurnal patterns of blood pressure, the ratios of day-to-nighttime average blood pressures were calculated for the final week of the studies (Fig. 1, E and F). All animals had a fall in daytime readings (12 h of light) relative to night (12 h of darkness) of between 3 and 10%. Both NaCl and fat caused a blunting in the fall in daytime relative to nighttime diastolic pressure (Fig. 1E). Systolic pressure (Fig. 1F) was affected in qualitatively the same manner, but these differences were not significant.
In Fig. 2, the delta MAP of both studies combined is graphed to assess the overall effects of CHO (A), salt (B), or added fat (C). Fat-supplemented diets increased delta MAP on day 28. No significant effect, when studies were combined, was seen for NaCl or fructose.
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-1 subunit of Na-K-ATPase or NHE3 were not affected by treatment. NaPi-2 was increased by both fructose and salt (Table 3). NKCC2, the bumetanide-sensitive Na-K-2Cl cotransporter, was decreased by fructose. NCC, the thiazide-sensitive NaCl cotransporter, was markedly decreased by NaCl, as was the -subunit of the ENaC. High salt increased band density for -ENaC and the 85-kDa band associated with -ENaC, whereas it decreased the 70-kDa band. -ENaC on immunoblots has two specific bands associated with it; a major band at 85 kDa, and a minor, more diffuse band(s) at 70 kDa (37). These bands have been shown to be regulated independently, for example, by aldosterone (37) and vasopressin (15), so they were independently evaluated for density.
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-ENaC was decreased by high NaCl in this study, and - and -ENaC (85-kDa band) were increased. However, unlike study 1, NCC and -ENaC (70-kDa band) were not decreased by high NaCl in this study. We could not directly evaluate the effect of added fat on these transport proteins, because these studies were done at different times and for immunoblotting analyses it is difficult to combine studies, because data are normalized by the control mean rather than presented as an absolute value.
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DISCUSSION |
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24% of the U.S. adult population (38) have been estimated to be afflicted with "metabolic syndrome" or "syndrome X," a clustering of features most likely centering around insulin resistance, including modest hypertension, hypertriglyceridemia, low HDL levels, microalbuminuria, and abdominal obesity (38). Hypertension, most commonly, is not caused by solely genetic defects but a complex interplay between environmental predisposition and genes. Thus, in these studies, we chose to study a normotensive, salt-resistant strain of rats and examine not only the development of increased blood pressure with a variety of dietary manipulations, but how the kidney may be able to protect against this rise. We began to decipher at the molecular level, with regard to regulation of renal sodium and water reabsorption, how and why these protective measures may fail over time. This will hopefully provide insight as to how to best prevent the initiation of blood pressure changes and its acceleration into severe hypertension and associated pathologies. Our studies, unlike several others (2, 5, 11, 12, 20, 22, 24, 47, 50), but in agreement with some (7, 10), suggest that fructose, as substituted for a more complex CHO, cornstarch, does not dramatically affect blood pressure in the male Sprague-Dawley rat. There may be multiple reasons for this, e.g., rat strain and age. Furthermore, the method of blood pressure measurement may have played a role. We used continuous radiotelemetry to record blood pressure, whereas most others used tail-cuff readings in conscious animals. Activation of the sympathetic nervous system while using tail-cuff blood pressure systems may affect outcome, especially in the fructose-fed model of hypertension, which has been shown to have elevated sympathetic activity associated with it (50). To address this, we also analyzed our data separately in the light vs. dark period (when we would expect more activity) but found no effect of fructose. However, we did uncover a significant effect of both high NaCl and fat to increase the day-to-night ratio in diastolic blood pressure. That is, blood pressure did not fall as much in the daylight period in rats on high-fat and -salt diets. Rats should normally have a small decrease in blood pressure during the light period, because they are relatively at rest. In humans, this "dip" is expected at night. "Nondippers" or blunted dippers are associated with a greater risk of cardiovascular diseases (34, 39). The mechanisms underlying this phenomenon are not clear, but may involve autonomic dysfunction (34).
In addition, in many studies the "fructose diets" had a greater amount of fat than the "control diets" and it was more saturated (43). In some cases, rats fed the "test" diets gained more weight likely due to greater palatability and caloric density of the diets (44). We found that rats fed a slightly higher level of fat by the addition of 5% butter (to a level of 21% of caloric intake vs. 12%) had an increase in blood pressure that was modest, yet exacerbated over time. This suggests that the kidney may not have been able to respond to the rise in blood pressure with natriuresis as effectively in this case, possibly due to blunted downregulation of renin, aldosterone, and aldosterone-regulated proteins.
Finally, we found that the higher NaCl diet caused a measurable and immediate increase in blood pressure of 5–10 mmHg in our rats. This has been reported by others using radiotelemetry (27), which is sensitive and objective enough to reliably uncover this modest effect.
The high-NaCl diets, in general, resulted in a significant decrease in the abundances of -ENaC, NCC, and the 70-kDa band associated with -ENaC. These proteins have been shown by Knepper and associates (6, 32, 37) to be increased in rat kidney either through feeding a low-NaCl diet, infusion of aldosterone, or ANG II. Thus the decreases in these proteins seen with high-NaCl diets were an appropriate adaptation to limit sodium reabsorption in the distal tubule (distal convoluted tubule through collecting duct). The downregulation of these proteins, especially NCC, was blunted in rats fed fat-supplemented diets. This may have contributed to the increased positive slope in MAP observed in these rats. In contrast, some proteins were increased in abundance by high-NaCl diets. Those included the major bands associated with - and -ENaC and the AQP2 and -3. All of these proteins have been shown to be increased by vasopressin and, moreover, vasopressin levels have been shown to be increased in some cases by feeding rats a high-NaCl diet (4, 13). Nevertheless, we did not observe any differences in plasma vasopressin levels between the high- and normal-NaCl-fed rats in our study.
On the other hand, the increase in - and -ENaC may be a direct effect of lower aldosterone levels in the high-NaCl-fed rats. Beutler et al. (6) demonstrated a spironolactone-sensitive decrease in these two bands in rats treated with candesartan (an antagonist of the ANG II type I receptor) fed low-NaCl diets (rats had high aldosterone levels). One possible molecular mechanism for these increases that has been proposed with regard to lithium-treated rats is that decreased trafficking of these ENaC subunits to the apical plasma membrane renders them less susceptible to degradation (40). Thus there would be higher protein abundance, but less active channels. Another possibility, at least with regard to -ENaC, that has been proposed (37) is that the major (85 kDa) band is proteolytically activated via cleavage into the lower (70 kDa) band. Thus an increase in the upper band might suggest less activating proteolytic cleavage with high NaCl. Thus it becomes apparent that ENaC activity as a result of an increase in exclusively - and -ENaC (85-kDa band) with high NaCl cannot be determined from these studies.
Several renal transport proteins were affected by dietary fructose. NaPi-2 was increased in abundance, whereas NKCC2 and AQP2 were decreased. Phosphorus reabsorption has been shown to be upregulated by insulin in opossum kidney cells (1). Thus, if these animals were somewhat insulin resistant, higher circulating insulin levels may have contributed to this increase. We were not able to show significant differences in final circulating plasma insulin levels in our rats; however, they were not fasted before death, which most likely affected the sensitivity of this measure of insulin resistance. We did however show a significant increase in triglyceride levels in fructose-fed rats, another measure of insulin resistance. Bergstra et al. (5) reported increased kidney size as well as nephrocalcinosis with high-fructose diets and attribute this to increased phosphorus reabsorption in the intestine.
The decrease in thick ascending limb NKCC2 abundance with high fructose potentially could contribute to maintenance of a normotensive state in our rats. Abnormal upregulation of NKCC2 abundance has been shown to be related to hypertension in the offspring of maternal rats exposed to low-protein diets (36). Major hormonal regulators of renal sodium transport, i.e., aldosterone and ANG II, do not appear to regulate NKCC2 abundance; however, vasopressin increases it (15). In addition, at least two studies (46, 49) including our own (46) suggest that increased NO production may increase its abundance. Nishimoto et al. (43) reported a decrease in renal medullary endothelial nitric oxide synthase in fructose-fed rats.
Finally, in both our studies, the fructose-fed rats had larger kidneys. The mechanisms underlying this increase in size, what anatomic features were affected, and whether they are physiologically relevant are not known. Renal function, including creatinine clearance, was not measurably different. Nevertheless, Manitius et al. (35) reported hyperfiltration, increased creatinine clearance, and hyperplasia of mesangial cells of the glomerulus with fructose feeding.
Overall, these studies suggest a blunting of the necessary and expected downregulation of the renin-angiotensin-aldosterone system, its regulated proteins, and fractional excretion of sodium with high NaCl in rats that are cosupplemented with added fat. This might be particularly relevant in influencing the modest rise in blood pressure observed with insulin resistance, which can often progress to more severe hypertension with renal function decline.
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GRANTS |
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FOOTNOTES |
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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.
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REFERENCES |
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-ENaC abundance in obese Zucker rats. Am J Physiol Renal Physiol 281: F639–F648, 2001., , and subunit proteins in rat kidney. J Clin Invest 104: R19–R23, 1999.[Medline]
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of fructose by 2-way ANOVA (CHO x NaCl level) where CHO is carbohydrate. E and F: effect of dietary treatments on the ratio of day-to-nighttime recorded diastolic and systolic blood pressures, respectively. High-fat-fed rats on high-NaCl diets had an increased day-to-night ratio of diastolic blood pressure. *Significant effect of high NaCl and 
of fat by 3-way ANOVA (CHO x NaCl x 
