SPR4-peptide induces a dramatic increase in fat mass/weight in HYP and WT mice.

<p>Dual Energy X-ray Absorptiometry (DEXA) measurements using a Lunar PIXImus system were carried out as described previously and discussed in methods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone.0097326-David2" target="_blank">[140]</a>. Measurements are shown for mice prior to pump implantation and after sacrifice 28 days later. The temporal percentage change measurements are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-g007" target="_blank">Figure 7</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t005" target="_blank">Table 5</a>. (<b>A</b>) <b><u>Percentage Fat Mass (% FAT Mass)</u></b>. HYPVE %-Fat-Mass was significantly less than WTVE %-Fat-Mass at all time-points. SPR4 peptide treatment significantly increased time-dependent gain in %-Fat-Mass for HYP mice (HYPSPR4) but not WT mice (WTSPR4) relative to respective vehicle groups. Following 2-way ANOVA analysis, phenotypic variation (including SPR4-treatment) was highly significant accounting for 31.86% of the total variance (F = 46.72, DF_n = 3, Df_d = 32 and <i>P<0.0001</i>). Also, time-changes were highly significant accounting for 53.69% of the total variance (F = 236.22, DF_n = 1, Df_d = 32 and <i>P<0.0001</i>). The phenotype/time <i><u>interaction</u></i> was also significant accounting for 7.18% of the total variance (F = 10.52, DF_n = 3, Df_d = 32 and <i>P<0.0001</i>). (<b>B</b>) <b><u>Total Weight (gm).</u></b> HYPVE-mice weight was significantly less than WTVE-mice weight at all time-points. SPR4 peptide treatment significantly decreased time-dependent gain in weight for both HYP mice (HYPSPR4) and WT mice (WTSPR4) relative to respective vehicle groups. Following 2-way ANOVA analysis, phenotypic variation (including SPR4-treatment) was highly significant accounting for 64.65% of the total variance (F = 40.6, DF_n = 3, Df_d = 32 and <i>P<0.0001</i>). Also, time-changes were highly significant accounting for 13.64% of the total variance (F = 25.69, DF_n = 1, Df_d = 32 and <i>P<0.0001</i>). The phenotype/time <i><u>interaction</u></i> was significant accounting for 4.73% of the total variance (F = 2.97, DF_n = 3, Df_d = 32 and <i>P = 0.0463</i>). (<b>C</b>) <b><u>Ratio of Fat mass/Weight (% Ratio).</u></b> No significant differences in fat-mass/weight ratios were observed between groups at 0 weeks (baseline, prior to pump implantation). In contrast, SPR4 peptide treatment significantly increased time-dependent gain in fat-Mass/weight ratio for both HYP mice (HYP-SPR4) and WT mice (WTSPR4) relative to respective vehicle groups. The gain in HYP-SPR4 fat-Mass/weight ratio was more marked and significantly greater than the WT-SPR4 mice. Following 2-way ANOVA analysis, phenotypic variation (including SPR4-treatment) was highly significant accounting for 30.60% of the total variance (F = 9.86, DF_n = 3, Df_d = 32 and <i>P<0.0001</i>). Also, time-changes were highly significant accounting for 17.14% of the total variance (F = 16.58, DF_n = 1, Df_d = 32 and <i>P = 0.0003</i>). The phenotype/time <i><u>interaction</u></i> was significant accounting for 19.17% of the total variance (F = 6.18, DF_n = 3, Df_d = 32 and <i>P = 0.002</i>). <b><i><u>Index</u></i></b><u>: </u><b>WTVE</b> =  wild type mice infused with vehicle (0.9% physiological saline); <b>HYPVE</b>  =  X-linked hypophosphatemic rickets mice infused with vehicle (0.9% physiological saline); <b>WTSPR4</b>  =  wild type mice infused SPR4-peptide; <b>HYPSPR4</b>  =  X-linked hypophosphatemic rickets mice infused SPR4-peptide.</p

Temporal changes in (Weight, fat mass and fat mass/weight ratio) as measured by Dual Energy X-ray Absorptiometry (DEXA) for wild type and HYP mice infused with vehicle or SPR4-peptide for 28 days (See <b>Figure 6</b> for static changes).

<p>The mean percentage change over the 28 days for each metric was calculated and the percentage differences between the groups plotted as a histogram (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t005" target="_blank"><b>Table 5</b></a>). Values are mean percentage differences and are significant (* = P<0.05) unless indicated by NS (unpaired t test confidence interval  = 95%). Column headings represent; <b>WT</b>  =  wild type mice, <b>HYP</b>  =  X-linked hypophosphatemic rickets mice, <b>SPR4</b>  =  infused SPR4-peptide and <b>Vehicle</b>  =  Saline infused. Histogram bars to the left of zero on the y-axis indicate down regulation and to the right up regulation. DEXA measurements using a Lunar PIXImus system were carried out as described previously and discussed in methods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone.0097326-David2" target="_blank">[140]</a>. <b><i><u>Index</u></i></b><u>: </u><b>Weight (Wt)</b>  =  percentage difference in weight change over 4 weeks; <b>Fat-Mass</b>  =  percentage difference in total fat-mass change over 4 weeks; <b>Fat/Wt Ratio</b>  =  percentage difference in total fat-mass/weight change over 4 weeks.</p

Immunohistochemistry of kidney sections confirm changes in protein expression for Na dependent phosphate co-transporter (NPT2A; Slc34a1).

<p>NPT2a protein-expression (purple-stain) in renal cortex sections is markedly decreased in HYP mice (compare photos 1 and 3). SPR4 peptide suppresses NPT2a expression in WT mice (compare photos 1 and 2) but increases NPT2a expression in HYP mice (compare photos 3 and 4). Staining is localized to proximal convoluted tubules with little glomerular staining. Magnifications are 20× and are from representative sections (matched regions).</p

Bone (femur) gene expression (mRNA) comparisons as measured by quantitative RT/PCR (qRT-PCR) for wild type (WT) and HYP mice infused with vehicle or SPR4-peptide for 28 days.

<p>Mice were sacrificed on day 28 and femurs collected for RNA purification as described in methods. Column headings represent; WT  =  wild type mice, HYP  =  X-linked hypophosphatemic rickets mice, SPR4  =  infused SPR4-peptide and Vehicle  =  Saline infused. For gene analysis mRNA was prepared from bone marrow stromal cell “<i>depleted</i>” femurs as detailed in methods. For qRT-PCR gene analysis fold differences in expression calculated by the Pfaffl method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone.0097326-Pfaffl1" target="_blank">[163]</a> were statistically analyzed for significance using the One Sample t-test and the Wilcoxon Signed rank-test with theoretical means set to 1. Results are significant (* = p<0.05) unless indicated by NS (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t004" target="_blank"><b>Table 4</b></a> for detailed statistics). <b><i><u>Index</u></i></b><u>: </u><b>FAM20C</b>  =  Family with sequence similarity 20, member C Kinase also known as DMP4; <b>ENPP1</b>  =  Ectonucleotide Pyrophosphatase Phosphodiesterase 1; <b>ESP</b>  =  Osteotesticular protein tyrosine (OST-PTP); <b>Plin-2</b>  =  Perlipin-2; phosphatase; <b>Cyclophilin</b>  =  peptidylprolyl isomerase A (cyclophilin A); <b>BGLAP</b>  =  Osteocalcin or Bone Gamma-Carboxyglutamate (gla) protein; <b>PHEX</b>  =  Phosphate-regulating gene with Homologies to Endopeptidases on the X chromosome; <b>GAPDH</b>  =  Glyceraldehyde 3-phosphate dehydrogenase; <b>VEGF</b>  =  Vascular Endothelial Growth factor; <b>DMP1</b>  =  Dentin Matrix Protein 1; <b>SOST</b>  =  Sclerostin; <b>MEPE</b>  =  Matrix Extracellular Phosphoglycoprotein with ASARM -motif; <b>FGF23</b>  =  Fibroblast Growth Factor 23; <b>NS</b>  =  not significant; <b>NA</b>  =  not applicable, PHEX mutated in HYP; * = P<0.05. Histogram bars to the left of zero on the axis indicate down regulation and to the right up regulation.</p

Table of primary antibodies used in the study:

<p>Table of primary antibodies used in the study:</p

Percentage difference serum chemistry comparisons between wild type (WT) and HYP mice and mice infused with vehicle or SPR4-peptide.

<p>For absolute measurements in tabulated form see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t001" target="_blank"><b>Table 1</b></a>. Mice were sacrificed on day 28 and sera prepared from 16 hour fasted mice housed in metabolic cages. Values are means of percentage difference and are significant (* = P<0.05) unless indicated by NS (unpaired t test, confidence interval  = 95%; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t002" target="_blank">Table 2</a> for absolute numbers). Column headings represent: <b>WT</b>  =  wild type mice; <b>HYP</b>  =  X-linked hypophosphatemic rickets mice; <b>SPR4</b>  =  infused SPR4-peptide; <b>Vehicle</b>  =  Saline infused; <b>NS</b>  =  not significant; <b>ND</b>  =  not done; * = P<0.05. Histogram bars to the left of zero on the axis indicate down regulation and to the right up regulation.</p

Table of primers used for quantitative RT-PCR (qRT-PCR).

<p>Table of primers used for quantitative RT-PCR (qRT-PCR).</p

Urine chemistry results and comparisons for wild type and HYP mice infused with vehicle or SPR4-peptide.

<p>Mice were sacrificed on day 28 and sera prepared from 16 hour fasted mice housed in metabolic cages. Column headings represent; WT  =  wild type mice, HYP  =  X-linked hypophosphatemic rickets mice, SPR4  =  infused SPR4-peptide and Vehicle  =  Saline infused. Values are means with ± SEM (N = 6). Unpaired t tests with confidence intervals of 95% were used for statistical analysis. Unpaired t tests with confidence intervals of 95% were used for statistical analysis (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-g002" target="_blank"><b>Figure 2</b></a>). Superscript letters (a, b or c) added to the calculated serum values indicate; a =  significantly different to WT vehicle (WT-VE) infused mice p<0.008, b =  significantly different to HYP vehicle (HYP-VE) infused mice p<0.002 and c =  significantly different to HYP SPR4-peptide (HYP-SPR4) infused mice p<0.02. ND =  Not done. The percentage changes between <i><u>WT-VE versus WT-SPR4</u></i>, <i><u>HYP-VE versus HYP-SPR4</u></i> and <i><u>WT-VE versus HYP-VE</u></i> are shown in columns 3, 6 and 7 as indicated in the headings. In these columns (3, 6 and 7), the numbers that are significantly different (p<0.01) are denoted by a superscript asterisk (*). <b><i><u>Index</u></i></b><u>:</u><b>FEP(%)</b>  =  Fractional Excretion of Phosphate, <b>Fe Ca (%)</b>  =  Fractional Excretion of Calcium.</p

Percentage difference urine chemistry comparisons between wild type (WT) and HYP mice and mice infused with vehicle or SPR4-peptide.

<p>For absolute measurements in tabulated form see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t002" target="_blank"><b>Table 2</b></a>. Mice were sacrificed on day 28 and urine collected from 16 hour fasted mice housed in metabolic cages. Values are means of percentage difference and are significant (* = P<0.05) unless indicated by NS (unpaired t test confidence, interval  = 95%; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t003" target="_blank">Table 3</a> for absolute numbers). Column headings represent; <b>WT</b>  =  wild type mice, <b>HYP</b>  =  X-linked hypophosphatemic rickets mice, <b>SPR4</b>  =  infused SPR4-peptide and <b>Vehicle</b>  =  Saline infused. Histogram bars to the left of zero on the axis indicate down regulation and to the right up regulation. <b><i><u>Index</u></i></b><u>: </u><b>FE Uric Acid</b>  =  percentage change fractional Excretion of uric acid; <b>creatinine clearance</b>  =  percentage change creatinine clearance; <b>ASARM/Cre</b>  =  percentage change in ASARM/creatinine; <b>Ca/Cre</b>  =  percentage change in calcium/creatinine ratio; <b>Fe Ca</b>  =  percentage change in the fractional excretion of calcium; <b>FEP</b>  =  percentage change in the fractional excretion of phosphate; <b>NS</b>  =  not significant; * = P<0.05. Histogram bars to the left of zero on the axis indicate down regulation and to the right up regulation.</p