Dietary fluoride,unlike bromide or iodide,counteracts phosphorus-induced nephrocalcinosis in female rats

Supplemental dietary F has been shown to counteract P-induced nephrocalcinosis in female rats. In order to obtain information as to the specificity of this F effect, the effect of other halogens, namely Br and I, on P-induced nephrocalcinosis was studied in weanling female rats. Supplemental dietary Br (5.24 mmol/kg of diet) and I (1.43 mmol/kg of diet) did not influence P-induced nephrocalcinosis, whereas F at equimolar dietary concentrations had marked antinephrocalcinogenic activity. The halogens were added to the diets in the form of KBr, KI, and NaF; the diets were balanced for the kations with Cl salts. The addition of KI to the diet to a concentration of 5.24 mmol/kg caused pronounced growth retardation, decreased feed intake, hepatomegaly, and signs of lethargy. It is concluded that the protective effect of dietary F against P-induced nephrocalcinosis does not extend to other halogens.     

Influence of diet on the development of nephrocalcinosis in the rat

A histological examination of the effect of a purified diet containing 20% alpha protein (an alkali-treated soyprotein) on the development of nephrocalcinosis induced by intraperitoneal injections of 0.5 neutral (pH 7.4) sodium phosphate was carried out in female weanling rats. Animals that were fed a standard commercial laboratory diet and given daily injections of phosphate for six or ten days developed a form of nephrocalcinosis that consisted mainly of intraluminal (intratubular) calcification at the junction of the outer and inner stripes of the outer medulla and in the inner stripe of the outer medulla. By contrast, rats that were fed the alpha protein diet and given injections of phosphate for six or ten days developed a form of nephrocalcinosis that was characterized primarily by a type of tubular basement membrane calcification at the junction of the inner stripe of the outer medulla and the inner medulla. The differences in nephrocalcinosis between the two dietary groups and the fact that an alpha protein diet by itself can cause renal calcification, leads to the suggestion that some component(s) or factor(s) in the alpha protein diet strongly influence(s) the development of nephrocalcinosis induced by injected neutral sodium phosphate.     

Diet-induced nephrocalcinosis and urinary excretion of albumin in female rats

This study was carried out to test the hypothesis that diet-induced nephrocalcinosis causes enhanced loss of albumin in urine, irrespective of the composition of the nephrocalcinogenic diet. Female rats were fed various purified diets for 28 days. There was a control diet (0.5% Ca, 0.04% Mg, 0.4% P, 15.1% protein, wt/wt), a low Mg (0.01% Mg), a high protein (30.2% protein) and a high P diet (0.6% P). The low Mg and high P diet induced nephrocalcinosis as demonstrated histologically and by markedly increased concentrations of kidney Ca. In rats fed the high protein diet, nephrocalcinosis was essentially absent. Group mean values of urinary excretion of albumin and plasma concentrations of urea were increased in rats fed either the low Mg or high P diet. The high protein diet did not affect urinary albumin but caused lysozymuria which was not seen in the other groups. Plasma urea was increased in rats fed the high protein diet. In individual rats, the concentration of Ca in kidney and urinary albumin excretion were positively correlated. It is suggested that nephrocalcinosis in female rats induced by either low Mg or high P intake causes kidney damage which in turn leads to increased concentrations of albumin in urine and urea in plasma.     

Vitamin D, within its range of fluctuation in commercial rat diets, does not influence nephrocalcinogenesis in female rats.

Massive, toxic doses of vitamin D have been shown to cause nephrocalcinosis in rats, but the effect of this vitamin within its range of fluctuation in commercial rat diets was unknown. Therefore, in two experiments with young female rats, the effect on nephrocalcinosis of a moderately increased level of vitamin D in the diet was studied, that is 5000 IU/kg versus the recommended concentration of 1000 IU/kg. This was done using purified diets with 0.5% (w/w) calcium and 0.04% magnesium containing either 0.2 or 0.6% phosphorus (P). Rats fed the diets containing 0.6% P showed severe kidney calcification compared to those fed the 0.2%-P diets. The level of vitamin D in the 0.2 and 0.6%-P diets did not affect kidney calcification. Bone density was increased after feeding diets containing 5000 instead of 1000 IU of vitamin D/kg. This study suggests that, within 28 days, a moderate increase of the amount of vitamin D in the diet has no influence on the development of kidney calcification. This in turn suggests that the variation in nephrocalcinosis severity and incidence seen in practice in rats fed different commercial diets is unlikely to be related to the different vitamin D concentrations in these diets. However, in rats fed such diets bone metabolism may be influenced differently.     

Phosphorus-induced nephrocalcinosis in female rats: a study on regression and clinical abnormalities

The question addressed was whether preestablished phosphorus (P)-induced nephrocalcinosis would regress after dietary P restriction. Female rats were fed purified diets containing either 0.2% (w/w) P (low P) or 0.6% P (high P). After 29 days, the high-P diet had caused massive nephrocalcinosis as demonstrated chemically (by the analysis of calcium in kidney) and histologically (by inspection of kidney sections stained for calcium phosphate deposits). Switching rats from the high P to the low P diet did not result in a decrease in the degree of nephrocalcinosis within 91 days. Thus, P-induced nephrocalcinosis may not regress upon subsequent P restriction. Rats that had been fed either the 0.2 or 0.6% P diet for 56 days were examined clinically with respect to 14 selected variables. None of the variables discriminated between rats with or without nephrocalcinosis. This might imply that P-induced nephrocalcinosis in female rats does not cause significant discomfort.     

Greater effect of dietary potassium tripolyphosphate than of potassium dihydrogenphosphate on the nephrocalcinosis and proximal tubular function in female rats from the intake of a high-phosphorus diet

We examined whether a difference in potassium dihydrogenphosphate (KH2PO4) and potassium tripolyphosphate (K5P3O10) as dietary phosphorus sources could differentially effect the nephrocalcinosis and proximal tubular function in female rats. Rats were fed on a diet containing KH2PO4 or K5P3O10, at the normal phosphorus level (normal phosphorus diet) or at a high phosphorus level (high-phosphorus diet) for 21 d. Nephrocalcinosis, as confirmed by a histological examination, was apparent in all rats fed on the high-phosphorus diet, and this condition was more severe in those rats fed on K5P3O10 than in those fed on KH2PO4. As indicators of the proximal tubular function, the N-acetyl-beta-D-glucosaminidase activity in urine and the urinary beta2-microglobulin excretion were significantly increased in those rats fed on the high-phosphorus diet containing K5P3O10. These results indicate that the intake of a high-phosphorus diet, more strongly influenced the nephrocalcinosis and proximal tubular function when K5P3O10 rather than KH2PO4 was used as the dietary phosphorus source.     

Studies on the mechanism of inhibition of 2-acetylaminofluorene toxicity by butylated hydroxytoluene.

Male rats were placed on a diet containing 0.05% (w/w) of the hepatic carcinogen 2-acetylaminofluorene (AAF). They ceased to gain weight. However, when the carcinogenic diet was supplemented with butylated hydroxytoluene (BHT) (0.5% w/w), an antioxidant, the animals gained weight at approximately one-half of the normal rate. This observation led to a series of experiments aimed at elucidating the mechanism(s) by which BHT reduced the toxicity of AAF. These initial studies were directed towards the effect of BHT on the extent and duration of the covalent binding of AAF with DNA. BHT feeding was shown to reduce the binding of carcinogen to hepatic DNA. Studies employing cells in culture demonstrated that BHT does not influence either excision repair or post-replication repair of DNA. These data indicate that a potential mechanism of action of BHT is at the anti-initiation level of carcinogen-induced DNA damage.     

Nutrition and kidney calcification in rats

Nephrocalcinosis is a 'spontaneous' disorder in rats which refers to the deposition of calcium salts in the kidney, preferably in the cortico-medullary region. Studies using defined, semi-purified diets have shown that low dietary concentrations of magnesium, high concentrations of calcium, high concentrations of phosphorus and low calcium: phosphorus ratios induce kidney calcification. Dietary phosphorus induced nephrocalcinosis in female rats is associated with increased kidney size and weight, tubular hyperplasia, fibrosis and increased excretion of albumin in urine. This suggests that nephrocalcinosis may impair kidney function. In rats fed different commercial diets the incidence of nephrocalcinosis can vary considerably. Differences in the degree of nephrocalcinosis in different experiments may negatively influence the comparability of experimental outcome, especially when this is affected by kidney function and structure. Experimental data are needed so that diets can be formulated that do not produce nephrocalcinosis without inducing other disorders.     

Mechanisms of butylated hydroxytoluene-mediated modulation of 2-acetylaminofluorene mutagenicity in rat and human hepatocyte/Salmonella assays

Salmonella typhimurium (TA98) mutagenesis assays were used to study the influence of the antioxidant butylated hydroxytoluene (BHT) on 2-acetylaminofluorene (2-AAF) mutagenesis, in search of the mechanism of the anticarcinogenic effects of BHT. Rats pre-treated with BHT in the diet (0.5% w/w for 10 days) provided hepatocytes and hepatocyte S9 which were more efficient in the activation of 2-AAF than were similar preparations from control rats. The increased release of mutagens from hepatocytes might explain the reported increase in the incidence of bladder tumours in BHT-treated rats. In contrast, the mutagenic activity of 2-AAF was inhibited by the in vitro addition of BHT into incubations where human or rat liver S9 and intact hepatocytes were used for metabolic activation. Both competitive and un-competitive inhibition by BHT of 7-ethoxycoumarin O-deethylation was observed in hepatocytes which suggested that the antimutagenic activity may be mediated by one or more mechanisms of cytochrome P-450 inhibition. BHT inhibition of the mutagenicity of N-OH 2-AAF and of rat urinary metabolites of 2-AAF indicated that effects other than those mediated by cytochrome P-450 also occur e.g. scavenging of reactive metabolites. It was concluded that BHT-modulation of 2-AAF metabolic activation and mutagenesis (which may relate to BHT-protection against hepatocarcinogenicity) involves multiple mechanisms.     

Sex-specific effects of spironolactone on blood pressure in gonadectomized male and female Wistar rats

A low-salt diet is known to decrease and salt excess to increase blood pressure in humans and rodents. Sex steroids seem to play a role in salt dependent hypertension. However, little is known about sex differences in mineralocorticoid receptor blockade between male and female rats. The objective of the work was at first to investigate the effects of a low-salt vs. a high-salt diet on blood pressure without the influence of gonadal steroids in male and female rats. Second, to determine the sex-specific effects of mineralocorticoid receptor blockade by spironolactone in high-salt and low-salt fed gonadectomized male and female animals. Normotensive male and female Wistar rats were gonadectomized and put on a low (NaCl<0.03%) or high (NaCl=4%) salt diet. On each diet animals received spironolactone or placebo. Blood pressure was measured by tail-cuff-method; 24-h urine samples were collected in metabolic cages and blood was collected for hormonal measurements. High-salt diet significantly increased systolic blood pressure in both sexes. This effect could be blocked effectively by spironolactone only in male rats. Spironolactone treatment significantly increased aldosterone levels in males and females independent of the sodium content of the diet. High sodium diet significantly increased relative kidney weight, which was not altered by spironolactone treatment. Independently of gonadal steroids a high-salt diet increased blood pressure in gonadectomized male and female rats. Spironolactone lowered blood pressure only in male not in female rats on a high-salt diet clearly indicating sex-specific effects of the mineralo-corticoid antagonist spironolactone.     

Pathological study on nephrocalcinosis in Fischer 344/Du Crj rats

Histopathological examinations on nephlocalcinosis of the Fischer 344 (F344) rats were carried out. As the results of comparison on its appearance among F344, Wistar and SD strains of rats, F344 female rats showed the most severe nephrocalcinosis. Nephrocalcinosis developed between 4 weeks and 8 weeks and was likely to keep its appearance through 108 weeks of the survival period of the rats. Histologically, mineral deposit was always observed at cortico-medullary junction. It seemed to locate at the outer portion of the basement membrane of the tubular epithelium, adjacent to the capillary wall in the connective tissue. Four weeks after ovariectomy at 4 weeks of age, the rats showed a decrease in degree of nephrocalcinosis. In contrary, the rats treated with estorone following ovariectomy revealed an increase in degree of nephrocalcinosis. It was suggested that the oestrogen-type sex hormone appeared to give a role in nephlocalcinosis.     

Greater Effect of Dietary Potassium Tripolyphoshate than of Potassium Dihydrogenphosphate on the Nephrocalcinosis and Proximal Tubular Function in Female Rats from the Intake of a High-phosphorus Diet

We examined whether a difference in potassium dihydrogenphosphate (KH₂PO₄) and potassium tripolyphosphate (K₅P₃O₁₀) as dietary phosphorus sources could differentially effect the nephrocalcinosis and proximal tubular function in female rats. Rats were fed on a diet containing KH₂PO₄ or K₅P₃O₁₀, at the normal phosphorus level (normal phosphorus diet) or at a high phosphorus level (high-phosphorus diet) for 21 d. Nephrocalcinosis, as confirmed by a histological examination, was apparent in all rats fed on the high-phosphorus diet, and this condition was more severe in those rats fed on K₅P₃O₁₀ than in those fed on KH₂PO₄. As indicators of the proximal tubular function, the N-acetyl-β-D-glucosaminidase activity in urine and the urinary β₂-microglobulin excretion were significantly increased in those rats fed on the high-phosphorus diet containing K₅P₃O₁₀. These results indicate that the intake of a high-phosphorus diet, more strongly influenced the nephrocalcinosis and proximal tubular function when K₅P₃O₁₀ rather than KH₂PO₄ was used as the dietary phosphorus source.     

Butylated hydroxytoluene is a ligand of urinary proteins derived from female mice

Mice secrete substantial amounts of protein, particularly proteins called the major urinary proteins (MUPs), in urine. One function of MUPs is to sequester volatile pheromone ligands, thereby delaying their release and providing a stable long-lasting signal. Previously, only MUPs isolated from male mice have been used to identify ligands. Here, we tested the hypothesis that MUPs derived from females may also sequester volatile organic compounds. We identified butylated hydroxytoluene (BHT), a synthetic antioxidant present in the laboratory rodent diet, as a major ligand bound to urinary proteins derived from C57BL/6J female urine. BHT was also bound to the male-derived proteins, but the binding was less prominent than that in female urine, even though males express approximately 4 times more proteins than females. We confirmed that the majority of BHT in female urine was associated with the high molecular weight fraction (>10 kDa) and the majority of the proteins that sequestered BHT were MUPs as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequestration of BHT by MUPs was further confirmed by employing the recombinant MUP8 whose natural analogue has been reported in both sexes. Therefore, our data indicate that MUPs expressed in both sexes can bind, transport, and excrete xenobiotics into urine and raise the possibility that in addition to the known role in chemical communication, MUPs function as a defense mechanism against exogenous toxins.     

Phophate-induced renal calcification in the rat.

Studies were done to investigate nephrocalcinosis produced in weanling female Wistar rats fed pelleted, semisynthetic diets. The rats were fed diets varying in concentrations of Ca and P supplied as inorganic salts for periods of 4--6 weeks and results compared with control rats fed laboratory rodent chow for the same period of time. Measurement of renal Ca and P concentrations showed that nephrocalcinosis was produced by semisynthetic diets with inorganic phosphate concentrations as low as 0.5% on a weight basis; in contrast, rats fed regular laboratory chow (P = 0.72%) showed no evidence of nephrocalcinosis. The severity of the lesion was proportional to dietary phosphate concentrations from 0.5 to 1.0% but other dietary factors modified the severity of the lesion. With the lower dietary phosphate of 0.5%, increasing dietary Ca from 0.5 to 1.0% decreased the severity of the renal calcification. Decreasing protein concentrations from 25 to 15% casein increased the severity of the renal lesions. Other dietary factors also appear to modify the phosphate-induced nephrocalcinosis since no lesions occurred in rats on laboratory chow. It is suggested that the availability of dietary phosphate may be a factor. The phosphate in the semisynthetic diets was totally inorganic while the natural foods of laboratory chow contain, at least in part, organic phosphate.     

Effects of gender on kidney function in magnesium-deficient rats

This study investigated the gender differences in the kidney function of magnesium (Mg)-deficient rats. Male and female rats were fed a control diet or a Mg-deficient diet for 21 d. Mg-deficient diet had no significant effect on kidney calcium (Ca) or phosphorus (P) concentration in male rats, while Ca and P concentrations in female rats were significantly higher in Mg-deficient rats than in the control rats. With regard to indicators of kidney function, no significant differences in creatinine clearance and serum urea nitrogen concentration were observed among the groups. Serum albumin concentrations were significantly lower in rats fed the Mg-deficient diet than in rats fed the control diet. In both sexes, urinary albumin excretion was significantly higher in rats fed the Mg-deficient diet than in rats fed the control diet. Gender differences had no significant influence on creatinine clearance, serum urea nitrogen concentration, serum albumin concentration and urinary albumin excretion. These results suggest that gender differences have no effect on kidney function in Mg-deficient rats under the condition used.     

In-vivo monitoring of corticomedullary nephrocalcinosis in rats using computerized tomography.

An in-vivo method, computerized tomography (CT), was used to monitor nephrocalcinosis in female rats. CT density data correlated well with renal Ca content measured by atomic absorption spectrophotometry. In-vivo CT measurements revealed that the severity of nephrocalcinosis may change spontaneously with time. Manifest calcifications may exhibit spontaneous regression and are probably affected positively by high dietary Mg, in contrast to increased Ca. It is concluded that CT is a suitable and reliable non-invasive in vivo method to follow up time-dependent alterations in kidney calcifications in rats.     

High-protein nutrition during pregnancy and lactation programs blood pressure, food efficiency, and body weight of the offspring in a sex-dependent manner

Maternal low-protein diet during pregnancy is a risk factor for cardiovascular disease of the offspring in later life. The impact of high-protein diet during pregnancy on the cardiovascular phenotype of the offspring, however, is still unknown. We examined the influence of a high-protein diet during pregnancy and lactation on the renal, hemodynamic, and metabolic phenotype of the F1 generation. Female Wistar rats were either fed a normal protein diet (20% protein: NP) or an isocaloric high-protein diet (40% protein: HP) throughout pregnancy and lactation. At weaning, the offspring were fed with standard diet, and they were allocated according to sex and maternal diet to four groups: normal-protein male (NPm, n = 25), normal-protein female (NPf, n = 19), high-protein male (HPm, n = 24), high-protein female (HPf, n = 29). During the experiment (22 wk), the animals were characterized by repeated measurement of body weight, food intake, blood pressure, glucose tolerance, energy expenditure, and kidney function. At the end of the study period histomorphological analyses of the kidneys and weight measurement of reproductive fat pads were conducted. There were no differences in birth weight between the study groups. No influence of maternal diet on energy expenditure, glucose tolerance, and plasma lipid levels was detected. Blood pressure and glomerulosclerosis were elevated in male offspring only, whereas female offspring were characterized by an increased food efficiency, higher body weight, and increased fat pads. Our study demonstrates that a high-protein diet during pregnancy and lactation in rats programs blood pressure, food efficiency, and body weight of the offspring in a sex-dependent manner.     

Effects of diet and exposure to hindlimb suspension on estrous cycling in Sprague-Dawley rats

Various factors can disrupt the female reproductive cycle resulting in subfertility. The primary objective of this study was to determine whether physiological changes associated with exposure to hypogravity disrupt reproductive cycles. The hindlimb suspension (HLS) model was used to simulate the major physiological effects of hypogravity in female Sprague-Dawley rats. Also, to determine whether diet may influence reproductive results, rats were fed purified American Institute of Nutrition (AIN)-93G or chow diet. Rats (n = 9-11/group) subjected to HLS had lengthened estrous cycles due to prolonged diestrus, indicating hypoestrogenism. Interestingly, HLS rats fed AIN-93G but not chow diet had significantly reduced time spent in estrus and decreased plasma estradiol. Attenuation of hypoestrogenism in the chow-fed rats suggested that diet provided an exogenous source of estrogen. The mechanism involved in the disruption of estrous cycling remains to be determined. HLS increased urinary corticosterone (CORT) levels during the initial 4 days of HLS, suggesting that physiological responses to acute stress may be a potential mechanism in the disruption of estrous cycles. Higher basal urinary CORT was observed in rats fed chow vs. AIN-93G diet. HLS resulted in increased urinary CORT. However, two-way ANOVA indicated a significant HLS effect (P < 0.001) but no effect of HLS x diet effect on urinary CORT levels, suggesting that estrogenic activity associated with the chow diet did not enhance the stress response. The results of this study indicate that HLS, diet, and the combination of HLS and diet influence estrous cycling. This has important implications for future reproductive success in the hypogravity environment of space.     

Commercial rodent diets and nephrocalcinosis in weanling female rats.

This study addresses the questions to what extent commercial rodent diets would induce nephrocalcinosis, and which dietary components would be responsible for inducing this condition. For this purpose, 10 commercial diets were analysed for selected components and fed to weanling female rats. On the basis of histological inspection of kidney sections, two diets were found to produce significant nephrocalcinosis. The condition could be considered relatively mild because concentrations of Ca in kidney tissue were not increased. There was considerable variation between the commercial diets in the (analysed) concentrations of Ca, P, Mg and protein as well as in the diet-induced urinary pH, urinary volume and caecal weight. Of these parameters, only the dietary Ca:P ratio and group mean urinary pH correlated significantly with the observed variation in group mean calcification scores, the relationships being negative. It is suggested that the Ca:P ratio of commercial rodent diets is an important determinant of nephrocalcinosis.     

Protective effects of estrogen on gender-specific development of diet-induced hypertension.

Dietary and humoral factors are thought to be involved in the development of hypertension. This study investigated the interaction between diet and gonadal hormone status in the development and reversibility of hypertension. Normal male and female and ovariectomized (OVX) female Fischer rats were placed on either a high-fat (primarily saturated), refined carbohydrate (sucrose) (HFS) or a low-fat, complex carbohydrate (LFCC) diet at 2 mo of age, and body weight and systolic blood pressure (BP) were measured. Male and OVX female rats were initially on the diets for 7 mo, whereas normal female rats were on the diets for 2 yr. After this initial phase, a group of rats from each of the normal HFS groups were converted to the LFCC diet for a period of 1 mo (males) and 2 mo (females). The OVX females were subcutaneously implanted with a 0.5-mg estradiol (E2) pellet for 1 mo. A significant rise in arterial BP occurred within 12 mo in female and only 2 mo in male rats on the HFS diet, exceeding 140 mmHg after 24 and 7 mo, respectively. Conversion from the HFS to the LFCC diet led to a normalization of BP in both female and male rats. HFS diet-induced hypertension was accelerated by OVX in female rats, approaching the pattern seen in male rats. The effect of OVX was completely reversed by E2 replacement. BP did not significantly change in any of the LFCC groups at any time point, and E2 replacement had no effect on BP in the OVX LFCC group. All HFS groups had significantly greater body weight, with differences occurring sooner in the male and OVX rats compared with the female rats. Diet modification resulted in a partial but significant reduction of body weight, but E2 replacement did not. These results demonstrate that long-term consumption of HFS diet induces hypertension in both genders and is reversible by diet modification. Hypertension is significantly delayed in females with functional ovaries. This protection is lost by OVX and restored by estrogen replacement. Thus hormone status contributes to the delayed onset of diet-induced hypertension in females compared with males.