Male Sprague-Dawley rats (220–270 g) were purchased from the Medical Experimental Animal Center at Sun Yat-sen University. All animal protocols were conformed to the Experimental Animal Management Regulations of Sun Yat-sen University. Rats were acclimated in metabolic cages for 3 days prior to the start of the study. Rats were randomly divided into three experimental groups (N = 5 per group): (1) control group, (2) HP group, or (3) HP + PRO20 group. Animals in HP and HP + PRO20 groups drank high phosphate fluid (5 × phosphate buffered saline, pH 7.4, [Pi] = 50 mM) for 24 h (Ide et al., 2016) and the control group drank tap water. Five days prior to HP treatment, osmotic minipump (2001, Alzet, United States) was implanted to deliver vehicle or PRO20 at 700 μg/kg/d as previously described (Wang et al., 2016, 2020). Twenty four-hour urine was collected using metabolic cages.

Plasma and urine creatinine was determined by the QuantiChrom™ Creatinine Assay Kit (DICT-500, BioAssay Syatems, United States). Plasma and urine sodium, potassium and chlorine levels were determined by the Sodium, Potassium and Chlorine Assay Kit, respectively (Nanjing Jiancheng Bioengineering Institute, China). Plasma and urine phosphorus and calcium levels were determined by the Micro Blood Phosphorus and Calcium Concentration Assay Kit, respectively (Solarbio life sciences, China). Plasma and urine soluble PRR (sPRR) levels were determined by the ELISA kit (27782, IBL, Japan). Plasma FGF23, PTH and 1,25(OH)₂D₃ concentrations were assayed using the ELISA kits (Cloud-Clone Corp., China). All of these ELISA assays were performed according to the manufacturer’s protocols.

Renal BBMs were isolated by double magnesium chloride (MgCl₂) precipitation as previously described (Gattineni et al., 2009) with minor modifications. After removal of the renal capsule, the renal cortex was isolated and homogenized in 2 ml of cold 2 × homogenization buffer (12 mM Tris pH 7.4, 300 mM mannitol, 5 mM EGTA). MgCl₂ was added to a final concentration of 12 mM and samples were incubated on ice for 15 min with occasional mixing. Then the aggregated membranes were removed by 15-min centrifugation at 3,000 g and 4°C, and the supernatant was then centrifuged for 30 min at 40,000 g and 4°C. The pellet was resuspended in 1 ml of 1 × cold homogenization buffer supplemented with 12 mM MgCl₂. After a second incubation and 15-min centrifugation at 3,000 g and 4°C and the supernatant was recovered and centrifuged at 40,000 g, 4°C, for 30 min. The BBM pellets were resuspended in RIPA buffer. All solutions were supplemented with protease inhibitors (1 mM PMSF).

Protein samples were fractionated on SDS-PAGE (30 μg/well) and transferred to a nitrocellulose membrane (Millipore). Immunoblots were incubated overnight at 4°C with primary antibodies including anti-ACE (1:1,000, GTX100923, GeneTex, United States), anti-AGT (1:1,000, GTX103824, GeneTex, United States), anti-renin (1:1,000, sc-133145, Santa, United States), anti-PRR (1:1,000, HPA003156, Sigma, United States), anti-Npt2a (1:1,000, A6742, Abclonal, China), anti-Npt2c (1:1,000, ab155986, Abcam, United Kingdom) or anti-β-actin (1:10,000, A1978, Sigma, United States) antibody in 1.5% (w/v) bovine serum albumin (BSA, Sigma, United States) in a TBS-T buffer [150 mM NaCl, 10 mM Tris (pH 7.4/HCl), 0.2% (v/v) Tween-20]. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:3,000, Thermo Fisher Scientific™ Pierce™). Specific signal was visualized by ECL kit (Thermo Fisher Scientific™ Pierce™). The protein bands were detected using Amersham Imager 600 and quantified by Image Pro Plus version 6.0 software (Molecular Dynamics).

Total RNA was extracted using Trizol (TRIzol, Invitrogen) following manufacturer’s instructions. RNA concentrations were quantified at 260 nm, and purity and integrity were determined using NanoDrop 2000. Reverse transcription was performed using iScript™ cDNA Synthesis Kit (Bio-Rad, United States). The mRNA expression was measured by quantitative RT-PCR using SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus, TaKaRa, China). The following primers were used: ACE: 5′-GAGCCATCCTTCCC–TTTTTC-3′ (forward) and 5′-CCACATGTTCCCTAGCAG-GT-3′ (reverse), AGT: 5′-AGCATCCTCCTTGAACTCCA-3′ (forward) and 5′-TGATTTT TGCCCAGGAT- -AGC-3′ (reverse), renin: 5′-GATCACCATG AAGGGG-GTCTCTGT-3′ (forward) and 5′-GTTCCTGAAG GGATTCTTTTGCAC-3′ (reverse), PRR: 5′-CTGGTGGCG- -GGTGCTTTAG-3′ (forward) and 5′-GCTACGTCTGGGAT-TC GATCT-3′ (reverse), Npt2a: 5′-GCCAGCATGACGTTTG TTGT-3′ (forward) and 5′-ATCACACCCAGG–CCAATGAG-3′ (reverse), Npt2c:5′-TGACTGTCCAAGCGT-CTGTC-3′ (forward) and 5′-TTCATCCCGATCCCCTGACT-3′ (reverse). GAPDH served as an internal control and its primer sequences were: 5′-GTCTTCACTACCA-TGGAGAAGG-3′ (forward) and 5′-TCATGGATGACCTT-GGCCAG-3′ (reverse).

Under anesthesia, kidneys were harvested and fixed with 10% paraformaldehyde. Paraffin embedded kidney sections were processed for IHC as previously described (Wang et al., 2015). Primary antibody for PRR (1:200, ab40790, Abcam, United Kingdom) was incubated overnight at 4°C. Sections were washed three times with 0.01 M PBS buffer and secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:300, Thermo Fisher Scientific) was incubated at room temperature for an hour. The staining procedure was performed using DAB Horseradish Peroxidase Color Development Kit (P0202, Beyotime Biotechnology, China) according to the manufacturer’s protocols. Immunohistochemical staining was detected with an Olympus BX 63 microscope (20 × and 40 × objective).

The MC3T3-E1 cells were obtained from as a gift from Dr. Zhi Tan (Sun Yat-sen University). Cells were cultured in MEM-αalpha (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, and 1% penicillin-streptomycin. Cells were seeded on 6 well plates. After 24 h, the cells were starved in media containing 0.5% FBS for 24 h. Then the cells were treated with 10 mM Pi for another 24 h. To evaluate the effects of PRR on the levels of FGF23, PRO20 was given at 10 nM. To further verify the involvement of PRR, PRR was silenced by transfecting the cells with siRNA against PRR. Scrambled siRNA served as a control. SiRNA for mouse PRR and control siRNA were purchased from Ruibo Biotech (Guangzhou, China). After the treatment, the medium was collected and assayed for sPRR or FGF23 assays (EK5626, SAB, United States).

Data is expressed as mean ± standard error (SEM). All data points were included for analyses. Samples sizes were determined based on similar previous studies. Statistical analysis for animal and cell cultures experiments was performed by means of one-way analysis of variance (ANOVA) for multiple-group comparison or Student’s t-test for two-group comparison. A p-value below 0.05 was considered statistically significant.

To test whether HP activated the RAS, we determined the levels of RAS components in urine and plasma from rats on normal Pi (NP) or HP intake using ELISA. The results showed that the levels angiotensinogen (AGT), renin, sPRR in urine and plasma from the HP group were significantly increased as compared with NP controls (Figure 1). By qRT-PCR, renal cortical mRNA expression of angiotensin-converting enzyme (ACE), AGT, renin, PRR were all increased in the HP group as compared with NP controls (Figure 2A). These results have been validated by Western blotting analysis. Of note, this analysis detected increases in the protein abundances of both PRR and sPRR in the kidney of HP rats (Figure 2B). By immunohistochemistry, PRR protein expression was elevated in the collecting duct by HP treatment (Figure 2C), a pattern consistent with intercalated cell labeling as reported previously (Wang et al., 2016).

SD rats drank tap water, HP fluid alone or in combination with PRO20 treatment. Basic physiological data is shown in Table 1. Fluid intake was less but urine output was higher in HP rats as compared with vehicle control. This was paralleled with increased 24-h urinary excretion of Na⁺, K⁺, and Cl^– induced by HP treatment. However, plasma creatinine and osmolality remained unchanged. None of these parameters were affected by PRO20.

To address the functional role of PRR in Pi homeostasis, we examined the effect of PRO20 on phosphaturic response to HP intake. HP intake induced a significant increase in urinary Pi excretion within 24 h and this increase was blunted by PRO20 treatment (Figure 3B). In parallel, HP intake elevated circulating FGF23 and PTH, both of which were nearly normalized by PRO20 treatment (Figures 4A,B). Despite reduced urinary Pi excretion, PRO20 treatment in HP rats did not affect plasma Pi concentration (Figure 3A). In a sharp contrast, plasma Ca²⁺ concentration (Figure 3C), urinary Ca²⁺ excretion (Figure 3D), or plasma 1,25(OH)₂D₃ (Figure 4C) were unaffected by HP intake or PRO20 treatment.

In a separate experiment, we examined the effect of PRO20 on several key parameters of Pi homeostasis in 7-wk-old male SD rats under basal condition (n = 5 per group). The data showed that PRO20 had no effect on urinary Pi excretion (PRO20 429.2 ± 16.8 vs. CTR 432.4 ± 17.8 μmol/24 h, p > 0.05), plasma Pi concentration (PRO20 2.89 ± 0.06 vs. CTR 2.90 ± 0.08, mmol/L, p > 0.05), plasma FGF-23 (PRO20 374.3 ± 15.4 vs. CTR 381.3 ± 10.2 pg/ml, p > 0.05), or urine volume (PRO20 9.75 ± 0.42 vs. CTR, 10.45 ± 0.85 ml, p > 0.05).

Downregulation of renal expression of sodium-phosphate cotransporters is a key determinant of phosphaturic response during HP intake (Murer et al., 1999; Hernando et al., 2001; Giral et al., 2009; Bourgeois et al., 2013; Forster et al., 2013; Zhuo et al., 2020). Therefore, we determined renal expression of Npt2a and Npt2c by both qRT-PCR and Western blotting analysis. In response to HP intake, renal cortical mRNA expression of Npt2a was significantly decreased, which was blunted by PRO20 treatment (Figure 5A). In contrast, the mRNA expression of Npt2c showed no significant changes in the three groups (Figure 5A). Meanwhile, we examined the abundance of sodium-phosphate cotransporters in the kidney BBM by Western blotting analysis. The protein abundance of Npt2a in BBM was downregulated by HP intake as compared with the NP control and this downregulation was prevented by PRO20 (Figure 5B). In contrast, no change was observed in protein abundance of Npt2c in BBM (Figure 5B).

The observation of suppressed circulating FGF23 concentration by PRO20 treatment during HP intake prompted us to speculate that the bone might be a potential site of PRR regulation of FGF23 release. To address this possibility, we conducted in vitro experiments using MC3T3-E1 cells, a mouse osteoblast cell line. The cells were exposed to normal or high Pi (10 mM Pi) for 24 h followed by examination of expression of FGF23 as well as PRR. qRT-PCR results showed that the expression of PRR and FGF23 mRNA was both significantly increased by HP treatment (Figure 6A). Consistent with this result, Western blotting analysis demonstrated significant elevations of protein abundance of full-length PRR (fPRR) and sPRR (Figure 6B). ELISA results showed that the concentrations of sPRR and FGF23 in the medium were significantly increased by HP treatment (Figures 6C,D).

Next, we examined the functional role of PRR in regulation of the production of FGF23 in the MC3T3-E1 cells by using PRO20. The cells were pretreated for 1 h with 10 μM PRO20 and then treated with 10 mM Pi for 24 h. By qRT-PCR, HP treatment increased the expression of FGF23 mRNA, and this increase was blunted by PRO20 (Figure 7A). This result was subsequently validated at protein level by ELISA (Figure 7B).

To further verify the above-mentioned results obtained with the pharmacological approach, we conducted independent experiments using siRNA approach to knockdown PRR. The efficacy of the gene knockdown was confirmed by qRT-PCR and Western blotting analysis (Figures 8A,B). PRR knockdown significantly blocked HP-induced FGF23 expression as assessed by qRT-PCR (Figure 8C) and ELISA (Figure 8D).