High-phosphorus Diet Induces Upregulation Of Bmp2 And Spp1 MRNA Expression in Rat Kidney

Hiroshi Matsuzaki, Kikue Mori, Shinichi Katsumata 

To clarify the mechanism of the development of nephrocalcinosis induced by a high-phosphorus (P) diet, we focused on bone-related proteins such as bone morphogenetic protein 2 and osteopontin. This study investigated the effects of a high-P diet on the mRNA expression of Bmp2 and Spp1 in the rat kidney. Wistar rats were divided into two groups and fed a control diet (control group) or a high-P diet (high-P group). Kidney calcium and P concentrations were significantly increased in the high-P group compared to the control group. The mRNA expression of both Bmp2 and Spp1 was significantly increased in the high-P group compared to the control group. These results indicate that a high-P diet induces upregulation of Bmp2 and Spp1 mRNA in the kidney.

Keywords: high-phosphorus diet, nephrocalcinosis, Bmp2, Spp1, kidney, rat

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Introduction

Nephrocalcinosis is a disorder characterized by calcium (Ca) deposits in the renal parenchyma. Dietary phosphorus (P), in particular, is a pivotal factor in the development of this disorder. Administration of a high-P diet has been shown to result in increased Ca concentration in the kidney and Ca deposits in the renal parenchyma1-6）. Although the effect of a high-P diet on the development of nephrocalcinosis has been the focus of ongoing study, little is known about the mechanism responsible for the development of this disorder. Thus, a timely resolution of this issue is needed.

We reported that upon histological examination using transmission electron microscopy, hydroxyapatite-like needle-shaped crystals were observed in the renal tubules of rats fed a high-P diet6, 7）. X-ray microanalysis indicated that Ca deposits induced by a high-P diet are mainly composed of Ca and P7）. These results revealed that Ca deposits induced by a high-P diet are a form of hydroxyapatite. Based on this finding, we postulate that the development of nephrocalcinosis is controlled by a mechanism similar to that in bone formation. Therefore, the present study focused on bone-associated proteins such as bone morphogenetic protein 2 (BMP2). BMP2 is an osteogenic factor that exhibits osteogenesis-inducing activity8, 9）.  It is noteworthy that BMP2 was identified as a factor inducing ectopic bone formation10, 11）.  Thus, we hypothesized that if BMP2 plays an important role in the development of nephrocalcinosis, the expression of this protein in the kidney would be upregulated by a high-P diet. The present study aimed to determine whether a high-P diet affects the mRNA expression of Bmp2 in the kidney.

Meanwhile, our previous study reported that a high-P diet elevates osteopontin (OPN) expression in renal tubules using immunohistochemical analysis, suggesting that OPN may be implicated in the development of Ca deposits induced by a high-P diet12）.  Therefore, we investigated the changes in Spp1 mRNA expression in the kidney as well.

The results of the present study may provide valuable insights into the mechanism responsible for the development of nephrocalcinosis induced by a high-P diet.

Materials and methods

Animals and diets.

The present study was approved by the Animal Use Committee of the Tokyo University of Agriculture, and all animals were maintained in accordance with the university's guidelines for the care and use of laboratory animals (Approval No. 300049).

It is important to note that female rats develop nephrocalcinosis more readily than male rats13, 14）.  In addition, we reported that a high-P diet increases OPN expression in renal tubules of female rats12）. The present study then used female rats as experimental animals. Four-week-old female Wistar rats (Clea Japan, Tokyo, Japan) were housed in individual stainless-steel wire-mesh cages.  During the experimental period, cages were located in a room with controlled lighting under a 12-h light: dark cycle (light, 0800-2000 h), the temperature of 22 ± 1℃, and relative humidity of 60% to 65%.

The composition of the experimental diets is shown in Table 1. Experimental diets were prepared in reference to the AIN-93G diet15). The experimental diets contained two different P concentrations (control diet, 0.3%; high-P diet, 1.5%). The P concentration in the high-P diet was set to 1.5% since our previous studies observed that a dietary P concentration of 1.5% induces

severe nephrocalcinosis6, 7, 12, 14）and high OPN expression12）. All experimental diets had the same concentration of Ca (0.5%). P and Ca concentrations, as determined by analysis of the experimental diets, are shown in Table 1. Prior to the start of the study, all rats were acclimatized for 7 days, during which time they were given free access to the control diet and deionized water. After the acclimatization period, the rats were randomly divided into two groups with similar mean body weights and fed either the control diet (control group) or the high-P diet (high-P group) for 14 days. Rats were given free access to the assigned experimental diets and deionized water throughout the experimental period. At the end of the experimental period, rats were euthanized by intraperitoneal injection of the anesthetic mixture (medetomidine, midazolam, and butorphanol), and blood and kidney samples were collected for analysis. Blood samples were centrifuged and supernatants were used as serum samples. Kidneys were removed and the renal capsule was discarded. The serum and kidney samples were stored at －80℃ until needed for analysis. The right kidney was used for quantitative analysis of Bmp2 and Spp1 mRNA, and the left kidney was used for the analysis of Ca and P concentrations.

Mineral analysis.

Samples of the experimental diets and kidneys were ashed at 550℃ for 48 h in a muffle furnace, and minerals were extracted in 1 mol/L of HCl for analysis. Ca concentrations in the experimental diets and kidneys were determined by atomic absorption spectrophotometry16）. P concentrations in the experimental diets and kidneys were analyzed using the method of Gomori17）.

Quantitative analysis of Bmp2 and Spp1 mRNA.

Total RNA was isolated from the homogenized kidney using TRIzol reagent (Life Technologies, CA, USA) according to the manufacturer’s specifications. The amount and purity of the RNA were assessed using a NanoDrop 2000c (Thermo Fisher Scientific, MA, USA). cDNA was synthesized using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Life Technologies) and then subjected to quantitative real-time RT- PCR using SYBR Select Master Mix (Life Technologies). Specific primers18-20）were used for the analysis of Bmp2 (forward, 5’-ACCGTGCTCAGCTTCCATCAC-3’; reverse, 5’-TTCCTGCATTTGTTCCCGAAA-3’), Spp1 (forward, 5’-TGAGACTGGCAGTGGTTTGC-3’; reverse, 5’-CCACTTTCACCGGGAGACA-3’), and Gapdh (forward, 5’-TGGGAAGCTGGTCATCAAC-3’; reverse, 5’-GCAT-CACCCCATTTGATGTT-3’). The real-time PCR reaction was performed using a StepOne Real-Time PCR System (Applied Biosystems, CA, USA), and mRNA expression levels were normalized to Gapdh mRNA levels. The value of the control group was considered to be 1.00.

Statistical analysis.

Results are presented as the means ± SEM for each group of 6 rats. After conducting an F-test to determine the homogeneity of variances, a Student’s t-test was used to determine significant differences between the two groups. If the homogeneity of variances was not equal, Welch’s t-test was used instead of the Student’s t-test. Differences were considered significant at p ＜ 0.05

Results

Bodyweight, food intake, and serum analysis.

Final body weight and food intake were significantly decreased in the high-P group compared to the control group (Table 2). Kidney weight and mineral concentrations. Kidney wet and dry weights were significantly increased in the high-P group compared to the control group (Table 3). Kidney Ca and P concentrations were also significantly increased in the high-P group compared to the control group.

Renal Bmp2 and Spp1 mRNA expression.

Bmp2 mRNA levels in the high-P group were significantly increased compared to the control group (Fig. 1A).  mRNA expression of Spp1 was also significantly increased in the high-P group compared to the control group (Fig. 1B).

Discussion

One of the well-known physiological effects of a high-P diet is the development of nephrocalcinosis. In the present study, kidney Ca and P concentrations were increased in the high-P group compared to the control group, which is consistent with the results of the previous studies1-6）.  Previous studies reported that histological scores of nephrocalcinosis were positively correlated with kidney Ca and P concentrations4, 21）. Thus, although no histological observation of the kidney was carried out in the present study, increased kidney Ca and P concentrations indicate that nephrocalcinosis was induced by the high-P diet.

Because Ca deposits induced by a high-P diet are generally thought to be a form of hydroxyapatite6, 7）, we hypothesized that the development of nephrocalcinosis is controlled by a mechanism similar to that of osteo genesis. Thus, we examined the effects of a high-P diet on Bmp2 mRNA expression in the kidney. The results

showed that renal Bmp2 mRNA expression was increased in the high-P group and that a high-P diet induces upregulation of Bmp2 mRNA expression in the kidney. This finding may help account for the mechanism of nephrocalcinosis development induced by a high-P diet. Although definitive evidence supporting our hypothesis is lacking, one possibility is that the expression of osteogenic transcription factors is elevated in calcified regions in the kidney. BMP2 induces osteoblast differentiation and bone formation by regulating osteogenic transcription factors such as Osterix, Runx2, and Msx28, 9）; thus, BMP2 may promote nephrocalcinosis via the regulation of such osteogenic transcription factors.  At present, the detailed role of BMP2 in the development of nephrocalcinosis remains unclear. In addition,  since only the Bmp2 mRNA expression was measured in this study, it is not known whether there is a difference in the BMP2 protein expression. However, our results suggest a possible mechanism by which alterations in BMP2 expression play a potent role in the pathogenesis of high-P diet-induced nephrocalcinosis.

The present study focused on Spp1 mRNA expression in addition to Bmp2 mRNA expression. Our previous study using immunohistochemical analysis indicated high levels of OPN expression in the renal tubules of rats fed a high-P diet12）. The present study found that  Spp1 mRNA expression was increased by approximately  10-fold in the high-P group. Results of the present and previous studies reveal that a high-P diet induces OPN  expression in the kidney. On the other hand, other researchers have shown that OPN as a stone matrix is an important factor in the formation of urinary stones22, 23）;  thus, our previous report suggested that OPN may have a role as a matrix component in the formation of  Ca deposits induced by a high-P diet12）. However, Paloian et al. recently reported that OPN plays a role in the prevention of high-P diet-induced nephrocalcinosis in  OPN KO mice24）. Namely, the physiological role of OPN  on high-P diet-induced nephrocalcinosis appears to be suppression rather than a promotion of calcification in the kidney. Moreover, previous reports are described that  OPN levels increase to offset further pathologic ectopic calcification in kidney and vasculature as OPN is upregulated at the site of pathologic calcification.

As mentioned above, the present study suggested the importance of BMP2 and OPN in high-P diet-induced nephrocalcinosis. It is noteworthy that the effects of  BMP2 and OPN on the development of nephrocalcinosis are thought to be regulated by different mechanisms.  These mechanistic differences may indicate that the underlying mechanism of nephrocalcinosis is complicated. 　The next point to consider is that growth inhibition may have affected Bmp2 and Spp1 mRNA expression in the kidney because the present study observed a reduction of final body weight in a high-P group. Observations in our previous12） and present studies may help account for this point. We previously reported increased OPN  protein expression in renal tubules of rats whose growth

was not inhibited by a high-P diet12）. Moreover, the present study showed growth inhibition in the high-P group, but high levels of Spp1 mRNA expression in the kidney. Based on these observations, the present study appears to have little effect of growth inhibition of Bmp2 and Spp1 mRNA expression. However, the details of the relationship between growth inhibition and Bmp2  and Spp1 gene expression remain unclear. Therefore,  further investigations are needed to elucidate this point.

In conclusion, to clarify the mechanism of the development of nephrocalcinosis induced by a high-P diet,  the present study determined the effects of a high-P diet on Bmp2 and Spp1 mRNA expression in the kidney.  Kidney Ca and P concentrations were significantly increased in the high-P group compared to the control group. Further, mRNA expression of Bmp2 and Spp1  was significantly increased in the high-P group compared to the control group. These results indicate that a high-P diet promotes upregulation of Bmp2 and Spp1 mRNA expression in the kidney.

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