Regular article
Changes in the Renal Copper Concentration in Rats as a Function of Phosphorus Intake
2022 Volume 2 Issue 2 Pages reg-1-reg-8
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2022 Volume 2 Issue 2 Pages reg-1-reg-8
We have found that the renal copper concentrations are markedly decreased in rats fed a low-phosphorus diet or administered phosphate binders. To clarify the relationship between phosphorus intake and renal copper concentration, we examined the renal copper concentrations in rats fed three types of diet with different phosphorus content. Eighteen 4-week-old male Wistar rats were divided into 3 groups and fed either a low phosphorus diet (phosphorus concentration, 0.15%), a control diet (phosphorus concentration, 0.3%), or a high phosphorus diet (phosphorus concentration, 0.6%) for 4 weeks. The serum phosphorus concentration reflected the phosphorus intake, with the highest values in the high phosphorus diet group, control group, and low phosphorus diet group, in that order. In the liver, the low phosphorus diet group had a significantly lower copper concentration than the other two groups, but the difference was not large. The kidney copper concentrations were remarkably different among the groups, with the highest values in the high phosphorus diet group, control group, and low phosphorus diet group, in that order. There were no differences in the iron, zinc, and manganese concentrations among the groups, except for a higher liver iron concentration in the low phosphorus diet group. These results indicate that the renal copper concentration increases or decreases specifically in response to changes in the phosphorus intake.
Phosphorus is an essential element in the formation of bone salts and cell membranes. It is also a material found in several nucleotides and high-energy phosphate compounds and is a major intracellular electrolyte. Thus, its excess or deficiency induces various health disorders [1]. However, the risk of specific dietary phosphorus deficiency is low because much of the phosphorus in the diet is protein bound [2]. In contrast, the high use of phosphate compounds in food additives may lead to excessive phosphorus intake [3].
Inadequate urinary excretion of phosphorus, such as in chronic renal failure, may increase the serum phosphorus level and cause ectopic calcification due to the binding of phosphate with calcium [4]. If this calcification occurs in the coronary or cerebral arteries, it can induce fatal ischemic disease. For this reason, patients with chronic renal failure should take phosphate binders to inhibit the absorption of phosphorus in the diet and prevent hyperphosphatemia [5].
Because phosphorus is bound to dietary proteins, it is difficult to prepare a severely low phosphorus diet. But a phosphate binder can be administered to experimental animals to induce a low phosphorus state [6]. We found that the administration of phosphate binders, such as lanthanum carbonate or iron citrate, to normal rats decreased the copper concentration in the kidney [7,8]. This decrease in the renal copper concentration was also observed in rats fed a mildly low phosphorus diet [9], suggesting that the low phosphorus state caused the decrease, not the effect of the metals used in the phosphate binders.
No studies have been found to explain the phenomenon of a low phosphorus state reducing the kidney copper levels. Nor has the interaction between phosphorus and copper been examined in the first place. In the present study, to clarify the relationship between phosphorus intake and renal copper concentration, the effect of high phosphorus feeding on the renal copper concentration was examined.
Animal feeding
The experimental protocol followed the Guide for the Care and Use of Experimental Animals issued by the Prime Minister’s Office of Japan and was reviewed and approved by the Animal Ethics Committee of Kansai University (Approval No. 2005).
Eighteen 4-week-old male Wistar rats (SHIMIZU Laboratory Supplies Co., Kyoto) were divided into 3 groups. One group (control group) was fed a basal AIN93G diet (control diet) with a phosphorus concentration of 0.3% [10], and the other two groups (low phosphorus group and high phosphorus group) were fed a low phosphorus diet with a phosphorus concentration of 0.15% and a high phosphorus diet with a phosphorus concentration of 0.6%, respectively. The composition of each diet is summarized in Table 1. The low phosphorus diet was prepared by replacing potassium dihydrogen phosphate in the AIN93G mineral mixture with potassium chloride, and the high phosphorus diet was prepared by adding sodium dihydrogen phosphate to the AIN93G diet. Sodium chloride was also added to the low phosphorus and control diets to equalize the sodium intake of each group. As a result, the chloride ion concentrations in the diets were highest in the low phosphorus diet, the control diet, and the high phosphorus diet, in that order.
During the feeding period, the animals ingested the diets and water (tap water) ad libitum. After feeding for 4 weeks, the livers, kidneys, and blood were collected under isoflurane (Fujifilm Wako Pure Chemical Co., Tokyo) anesthesia. Some of the blood was centrifuged at 1,500 x g for 15 minutes to obtain serum. The livers and kidneys were frozen in liquid nitrogen and stored at –30˚C until analysis.
| Low P diet | Control diet | High P diet | |
|---|---|---|---|
| Ingredients (g/kg) | |||
| Casein | 200 | 200 | 200 |
| Sucrose | 100 | 100 | 100 |
| Soybean oil | 70 | 70 | 70 |
| Corn starch | 391.84 | 391.84 | 385.88 |
| Gelatinized corn starch | 132 | 132 | 132 |
| Phosphorus-free mineral mixture1) | 35 | – | – |
| AIN93G mineral mixture | – | 35 | 35 |
| AIN93G vitamin mixture | 10 | 10 | 10 |
| Cellulose | 50 | 50 | 50 |
| Choline bitartrate | 2.5 | 2.5 | 2.5 |
| L-Cystine | 3 | 3 | 3 |
| NaH2PO4 | – | – | 11.62 |
| NaCl2) | 5.66 | 5.66 | – |
| Phosphorus content (%)3) | 0.15 | 0.30 | 0.60 |
| Chloride ion (Cl–) content (%) | 0.68 | 0.50 | 0.16 |
1) Prepared by removing KH2PO4 from the AIN93G mineral mix and adding KCl to equalize the potassium concentration.
2) Added to equalize the sodium concentration.
3) Contains phosphorus derived from casein.
One kidney and approximately 1 g of liver were heated with 5 mL nitric acid until there were no solids. The obtained solution was diluted with pure water and filtrated through a 0.45 µm filter, and zinc, iron, copper, and manganese were determined using an atomic absorption spectrophotometer (AA-7000, Shimadzu, Kyoto) or inductively coupled plasma mass spectrometer (ICPMS-2030, Shimadzu, Kyoto). In the analysis with ICPMS, 45scandium was used as an internal standard. The other kidney was homogenized in 10 times volume of saline and centrifuged (105,000 x g, 60 minutes) to separate the soluble fraction and precipitate, and the respective copper concentrations were determined. Serum zinc and copper were measured using a commercial kit (Metalloassay LS, Metallogenics Co., Chiba).
Whole blood hemoglobin and serum total protein, triacylglycerol, total cholesterol, urea nitrogen, uric acid, chloride ion, calcium, magnesium, inorganic phosphorus, iron and total iron binding capacity were determined by Japan Medical Laboratory Co. (Kaizuka).
For each measurement, the differences among the groups were tested by one-way ANOVA followed by the Tukey’s multiple comparison test. GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego) was used as the statistical analysis application.
The body weights and organ weights at the end of the feeding period, as well as water consumption and feed efficiency during the feeding period are summarized in Table 2. In the low phosphorus group, body weight and feed efficiency were lower than in the other two groups. However, this group had a higher kidney weight per body weight and drank more water during the feeding period. Although the exact amounts were not measured, the low phosphorus group also had a higher urine output. On the other hand, the high phosphorus group did not differ from the control group in terms of the body and organ weights.
The serum biochemical parameters and hemoglobin concentrations are shown in Table 3. The serum inorganic phosphorus concentrations reflected the dietary phosphorus concentrations and were highest in the high phosphorus group, control group, and low phosphorus group, in that order. No differences were observed between the high phosphorus group and the control group for the other parameters. On the other hand, the low phosphorus group had lower triacylglycerols and higher uric acid, calcium, and hemoglobin values than the other two groups. Serum copper also tended to be lower in the low phosphorus group, but the difference was not significant.
The iron, zinc, copper, and manganese concentrations in the liver and kidneys are shown in Table 4. In the liver, a significant accumulation of iron was observed in the low phosphorus group. The copper concentrations tended to be higher with a higher phosphorus intake, with significant differences between the high and low phosphorus groups. In the kidneys, on the other hand, there were notable differences in the copper concentrations among the groups. That is, the low phosphorus group had the lowest kidney copper concentration, while the high phosphorus group had significantly higher values than the control group. In other words, the kidney copper concentrations were lower when the phosphorus intake was low and higher when the phosphorus intake was high.
The kidneys were divided into soluble and insoluble fractions, and their respective copper concentrations were measured. As shown in Figure 1, in both fractions, the copper concentrations were significantly higher with increasing phosphorus intake.
| Low P | Control | High P | ANOVA | |
|---|---|---|---|---|
| Body weight (g) | 245.0 ± 8.1a | 291.1 ± 3.1b | 295.8 ± 4.0b | p<0.001 |
| Feed efficiency1) | 0.411 ± 0.018a | 0.507 ± 0.009b | 0.473 ± 0.008b | p<0.001 |
| Water intake (mL/d) | 41.7 ± 2.6b | 36.3 ± 2.5ab | 31.6 ± 2.7a | p=0.047 |
| Liver weight (g/100 g body weight) | 4.09 ± 0.14 | 4.05 ± 0.06 | 4.21 ± 0.10 | NS2) |
| Kidney weight (g/100 g body weight) | 0.82 ± 0.02b | 0.74 ± 0.01a | 0.71 ± 0.01a | p<0.001 |
Values are means ± SEM (n=6). Tukey’s multiple comparison was performed when ANOVA was significant (p<0.05); means in the same row not sharing a common superscript differ significantly (p<0.05) in the multiple comparison.
1) Calculated from the formula: (body weight gain during the feeding period (g)) / (total food intake (g)).
2) NS: not significant
| Low P | Control | High P | ANOVA | |
|---|---|---|---|---|
| Serum biochemical parameters | ||||
| Total protein (mg/dL) | 5.3 ± 0.1 | 5.3 ± 0.1 | 5.4 ± 0.1 | NS1) |
| Triacylglycerol (mg/dL) | 52 ± 5a | 88 ± 16b | 73 ± 15b | p=0.002 |
| Total cholesterol (mg/dL) | 76 ± 4 | 75 ± 5 | 79 ± 4 | NS |
| Urea nitrogen (mg/dL) | 21 ± 2 | 19 ± 1 | 18 ± 1 | NS |
| Uric acid (mg/dL) | 1.23 ± 0.06b | 0.78 ± 0.09a | 0.77 ± 0.05a | p<0.001 |
| Chloride ion (mEq/L) | 100 ± 1 | 101 ± 1 | 100 ± 1 | NS |
| Calcium (mg/dL) | 12.55 ± 0.21b | 10.65 ± 0.14a | 10.85 ± 0.06a | p<0.001 |
| Magnesium (mg/dL) | 1.82 ± 0.05 | 1.87 ± 0.03 | 1.85 ± 0.06 | NS |
| Inorganic phosphorus (mg/dL) | 4.53 ± 0.37a | 7.02 ± 0.30b | 8.13 ± 0.20c | p<0.001 |
| Iron (µg/dL) | 289 ± 18 | 288 ± 16 | 269 ± 23 | NS |
| Transferrin saturation (%) | 62.4 ± 5.2 | 58.2 ± 3.4 | 50.7 ± 4.3 | NS |
| Zinc (µg/dL) | 237 ± 11 | 231 ± 13 | 226 ± 19 | NS |
| Copper (µg/dL) | 119 ± 9 | 155 ± 18 | 152 ± 27 | NS |
| Hemoglobin (g/dL) | 14.0 ± 0.2b | 13.5 ± 0.1a | 13.5 ± 0.1a | p=0.007 |
| Hematocrit value (%) | 40.8 ± 0.6 | 39.9 ± 0.3 | 39.5 ± 0.3 | NS |
Values are means ± SEM (n=6). Tukey’s multiple comparison was performed when ANOVA was significant (p<0.05); means in the same row not sharing a common superscript differ significantly (p<0.05) in the multiple comparison.
1) NS: not significant
| Low P | Control | High P | ANOVA | |
|---|---|---|---|---|
| Liver | ||||
| Iron | 98.8 ± 5.5b | 81.5 ± 3.1a | 83.4 ± 3.6ab | p=0.020 |
| Zinc | 27.0 ± 0.5 | 26.9 ± 0.6 | 27.2 ± 0.9 | NS1) |
| Copper | 3.29 ± 0.05a | 3.44 ± 0.05ab | 3.53 ± 0.07b | p=0.030 |
| Manganese | 2.79 ± 0.08 | 2.94 ± 0.31 | 2.44 ± 0.15 | NS |
| Kidney | ||||
| Iron | 45.9 ± 1.6 | 46.9 ± 1.2 | 47.9 ± 0.6 | NS |
| Zinc | 19.7 ± 1.3 | 21.4 ± 1.1 | 21.3 ± 0.7 | NS |
| Copper | 4.26 ± 0.13a | 12.19 ± 0.45b | 17.75 ± 1.21c | p<0.001 |
| Manganese | 1.07 ± 0.05 | 1.18 ± 0.09 | 0.99 ± 0.03 | NS |
Values are means ± SEM (n=6). Tukey’s multiple comparison was performed when ANOVA was significant (p<0.05); means in the same row not sharing a common superscript differ significantly (p<0.05) in the multiple comparison.
1) NS: not significant
The height of the box and the length of the vertical line show the mean and SEM (n=6), respectively. Means in the same frame not sharing a common superscript differ significantly (p<0.05) in the Tukey’s multiple comparison.
In the present experiment, potassium dihydrogen phosphate was replaced with potassium chloride in the mineral mixture for the preparation of the low phosphorus diet. Because sodium dihydrogen phosphate was used to prepare the high phosphorus diet, sodium chloride was added to the control and low phosphorus diets to equalize the sodium intake. As a result, the concentration of chloride ions in the diets was in the following order: low phosphorus diet > control diet > high phosphorus diet (Table 1).
Recently, European Food Safety Authority (EFSA) has calculated the Dietary Reference Values (DRV) for chloride from the DRV for sodium, based on the assumption that chloride ions are consumed as sodium chloride [11]. This EFSA document does not specify the effects of excessive intake of chloride ions alone, independent of sodium or potassium. Since very little chloride are excreted in the feces [12] and are mostly absorbed in the gastrointestinal tract [11], it is possible that the blood concentration of chloride ions is maintained at a constant level by increasing urinary excretion rather than reducing absorption.
The water consumption and kidney weights of the rats fed the low phosphorus diet were significantly higher than those of the other two groups (Table 2). Although the exact amounts were not measured, it was observed that this group also produced more urine than the other two groups. On the other hand, there was no difference in the serum chloride ion concentrations among the three groups (Table 3) despite the large difference in chloride ion intake. These results suggest that rats in the low phosphorus group drank large amounts of water to excrete excess chloride ions in the urine, resulting in the accumulation of more water in the kidneys and an increase in their weight.
The main focus of the current experiment was to determine whether the changes in the kidney copper concentration that occurred in rats on the low-phosphorus diet were also observed on the high-phosphorus diet; in other words, whether increases or decreases in the phosphorus intake led directly to increases or decreases in the kidney copper concentrations. The results, as shown in Table 4, indicate that the renal copper concentrations do indeed increase or decrease in dependence on phosphorus intake. That is, the renal copper concentrations decreased when the phosphorus intake was low and increased when the phosphorus intake was high. To date, there have been no reported cases of such a phenomenon.
The body weights were lower in the low phosphorus diet group than in the other two groups. Feed efficiency was also decreased, suggesting that energy metabolism was not sufficiently maintained due to phosphorus deficiency at a dietary concentration of 0.15%. It has been reported that rats fed a low-phosphorus diet (0.2% phosphorus) show decreased serum phosphorus concentration, decreased bone mineralization rate, and growth inhibition [13]. The growth inhibition observed in rats fed a low-phosphorus diet (0.15%) in this study may be due to delayed bone formation resulting from decreased bone mineralization rate, in addition to decreased energy metabolism caused by phosphorus deficiency.
We have observed a decrease in the renal copper concentration in 8-week-old rats on a low-phosphorus diet or phosphate-binder without changes in the body weight or kidney weight [7-9]. Therefore, it is unlikely that the decrease in the renal copper concentration observed in rats on a low phosphorus diet is a secondary effect of growth inhibition.
On the other hand, in an experiment in which rats were given excess phosphorus, renal hypertrophy and calcification were observed when the dietary concentrations exceed about 1% [14]. Since no such abnormalities were observed at the 0.6% dietary phosphorus concentration in the present study, it can be concluded that no damage occurred as a result of excessive phosphorus intake. Thus, the increase or decrease in the renal copper concentration with changes in the phosphorus intake appears to be a primary effect of the increase or decrease in phosphorus intake.
Thus, in the present experiment, the increase in renal copper concentration associated with a high phosphorus diet was not particularly damaging to renal function. However, since copper accumulation in tissues can cause tissue damage via the Fenton reaction, the health effects of copper accumulation at higher doses of phosphorus than in the present study should be examined. In addition, the effects of decreased renal copper concentrations due to a low-phosphorus diet on the physiological functions of copper, such as the activity of copper-containing enzymes, should also be examined.
The liver copper levels were significantly lower only when fed a low phosphorus diet (Table 4). The serum copper concentrations also tended to decrease in the low phosphorus group, although not significantly (Table 3). Although these changes are minor compared to the renal concentrations, they may indicate a change in whole-body copper dynamics as phosphorus intake increases or decreases.
Phosphorus homeostasis involves intestinal absorption, bone absorption and formation, renal excretion, and renal tubular reabsorption. These are regulated by the parathyroid hormone and 1,25-dihydroxy vitamin D [15]. A marked decrease in the urinary excretion of phosphorus was observed in rats with hypophosphatemia induced by the administration of phosphate-binders [7,8], suggesting that renal reabsorption of phosphorus may play an important role in phosphorus homeostasis. Phosphorus reabsorption in the renal tubules is regulated by fibroblast growth factor 23 (FGF23), and an increase in the serum phosphorus concentration increases the serum FGF23 concentration, while a decrease in the phosphorus concentration decreases the FGF23 concentration [16]. Such a change in the FGF23 concentration in response to an increase or decrease in the serum phosphorus concentration may have influenced the increase or decrease in the renal copper concentration. However, the relationship between FGF23 and urinary copper excretion is unknown. In addition, urine is not a major route of copper excretion, as copper is excreted via bile. Therefore, it is unlikely that FGF23 is involved in changes in the renal copper concentration.
Phosphate ions form insoluble salts with many metal ions. The significantly higher serum calcium concentration and the significantly higher liver iron and hemoglobin concentrations in the low phosphorus group (Table 3 and 4) may indicate that the low phosphorus diet decreased the phosphate concentration in the gastrointestinal tract and increased the amounts of soluble calcium and iron that were absorbed.
Copper ions will also form insoluble salts with phosphate ions. If such insoluble salts are formed in the blood, they may not be filtered by the glomeruli of the kidney and may accumulate in the kidney. However, when the kidneys were divided into soluble and insoluble fractions and the copper concentrations were measured, they both changed in response to phosphorus intake (Figure 1). Furthermore, such insoluble salts could also be formed between iron or zinc ions and phosphate ions, but only copper showed significant changes in the kidney concentration. In other words, it is difficult to establish a mechanism by which insoluble salts of phosphate and copper were formed in the blood and accumulated in the kidneys.
As described above, changes in phosphorus intake may affect calcium and iron absorption. In particular, there have been several reports on the interaction between iron and copper and excess iron above the required level has been reported to inhibit copper absorption [17,18]. Accordingly, the present results may be mediated by changes in iron absorption rather than a direct effect of phosphorus. Therefore, it is necessary to reexamine copper concentrations in various organs when iron doses are varied, including changes in phosphorus in the body.
This study was supported by Grant-in-Aid for Scientific Research (22K055180).
The authors have no conflicts of interest directly relevant to the content of this article.
