Renal Proteome in Mice with Different Susceptibilities to Fluorosis

<div><p>A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis due to their genetic backgrounds. They also differ with respect to several features of fluoride (F) metabolism and metabolic handling of water. This study was done to determine whether differences in F metabolism could be explained by diversities in the profile of protein expression in kidneys. Weanling, male A/J mice (susceptible to dental fluorosis, n = 18) and 129P3/J mice (resistant, n = 18) were housed in pairs and assigned to three groups given low-F food and drinking water containing 0, 10 or 50 ppm [F] for 7 weeks. Renal proteome profiles were examined using 2D-PAGE and LC-MS/MS. Quantitative intensity analysis detected between A/J and 129P3/J strains 122, 126 and 134 spots differentially expressed in the groups receiving 0, 10 and 50 ppmF, respectively. From these, 25, 30 and 32, respectively, were successfully identified. Most of the proteins were related to metabolic and cellular processes, followed by response to stimuli, development and regulation of cellular processes. In F-treated groups, PDZK-1, a protein involved in the regulation of renal tubular reabsorption capacity was down-modulated in the kidney of 129P3/J mice. A/J and 129P3/J mice exhibited 11 and 3 exclusive proteins, respectively, regardless of F exposure. In conclusion, proteomic analysis was able to identify proteins potentially involved in metabolic handling of F and water that are differentially expressed or even not expressed in the strains evaluated. This can contribute to understanding the molecular mechanisms underlying genetic susceptibility to dental fluorosis, by indicating key-proteins that should be better addressed in future studies.</p> </div

Expression of differentially significant kidney proteins between control A/J <i>vs</i> control 129P3/J mice.

a<p>Experimental molecular weight (kDa)/p<i>I</i> of protein spot in the gel (Mean of min. and max.) based on the coordinates of landmark proteins. <i>^b</i>Theoretical molecular weight (kDa)/p<i>I</i> of theoretical protein. <i>^c</i>Number of peptides identified and score. <i>^d</i>Differences in expression in relation to 129P3/J mice (↓ down-modulation; ↑ up-modulation); Individual <i>P</i> value after ANOVA. <i>^e</i>Identification is based on protein ID from IPI (international protein index) protein database (<a href="http://www.uniprot.org/" target="_blank">http://www.uniprot.org/</a>). <i>^f</i>Category of protein based on its primary biological function according to Rison (2000) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053261#pone.0053261-Rison1" target="_blank">[18]</a>.</p

Venn diagram showing distribution of total kidney proteins identified with differences in expression from the 2D-PAGE and LC-MS/MS-based proteome.

<p>The numbers indicate the total protein identified from each comparison (control, 10 and 50 ppmF A/J and 129P3/J) and the number of proteins commonly identified between them.</p

Expression of differentially significant kidney proteins between 10 ppmF A/J <i>vs</i> 10 ppmF 129P3/J mice.

a<p>Experimental molecular weight (kDa)/p<i>I</i> of protein spot in the gel (Mean of min. and max.) based on the coordinates of landmark proteins. <i>^b</i>Theoretical molecular weight (kDa)/p<i>I</i> of theoretical protein. <i>^c</i>Number of peptides identified and score. <i>^d</i>Differences in expression in relation to 129P3/J mice (↓ down-modulation; ↑ up-modulation); individual <i>P</i> value after ANOVA. <i>^e</i>Identification is based on protein ID from IPI (international protein index) protein database (<a href="http://www.uniprot.org/" target="_blank">http://www.uniprot.org/</a>). <i>^f</i>Category of protein based on its primary biological function according to Rison (2000) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053261#pone.0053261-Rison1" target="_blank">[18]</a>.</p

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BMD and histomorphometric parameters in trabecular region of tibiae from A/J and 129P3/J mice treated with 0, 10 or 50 ppm F in drinking water for 8 weeks.

<p>Values are mean ± SD, n = 8/group. A =  Bone mineral density (BMD); B =  Specific bone surface (BS/BV); C =  Bone volume fraction (BV/TV); D =  Bone surface density (BS/TV); E =  Trabecular separation (Tb.Sp); F =  Trabecular thickness (Tb.Th); G =  Trabecular number (Tb.N); H =  Trabecular bone pattern factor (Tb.Pf). *<i>p</i><0.05, **<i>p</i><0.01 and *** <i>p</i><0.001 represents significant differences between strains, for each group. No statisticallly significant differences were found for each strain after receiving F.</p

Schemes of the protein-based mechanisms triggered by F in osteoblasts and osteoclasts.

<p>Identified proteins with greater abundance in 129P3/J in comparison to A/J related to bone metabolism are shown in red. (A) Exportin enhances transport of cfab/Runx-2 to the nucleus activating transcription of collagen type I genes. Cadherins promote intercellular adhesion enhancing osteoblast differentiation. Nox-mediated ROS production enhances RANK ligand (RANKL) that interacts to its receptor (RANK) to induce osteoclast differentiation. (B) The treatment of 129P3/J with high F promotes enhancement of Nox, CDH, cadherin, catetin and SHIP while (C) in A/J, F at high dose promotes enhancement of SHIP and reduction of exportin.</p

Expression of unique kidney proteins between A/J and 129P3/J mice.

a<p>Experimental molecular weight (kDa)/p<i>I</i> of protein spot in the gel (Mean of min. and max.) based on the coordinates of landmark proteins. <i>^b</i>Theoretical molecular weight (kDa)/p<i>I</i> of theoretical protein. <i>^c</i>Number of peptides identified and score. <i>^d</i>Identification is based on protein ID from IPI (international protein index) protein database (<a href="http://www.uniprot.org/" target="_blank">http://www.uniprot.org/</a>). <i>^e</i>Category of protein based on its primary biological function according to Rison (2000) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053261#pone.0053261-Rison1" target="_blank">[18]</a>.</p

BMD and histomorphometric parameters in cortical region of femur from A/J and 129P3/J mice treated with 0, 10 or 50 ppm F.

<p>Values are mean ± SD, n = 8/group. A =  Bone mineral density (BMD); B =  Mean total cross-sectional tissue area (T.Ar); C =  Mean total cross-sectional bone area (B.Ar); D =  Mean total cross-sectional tissue perimeter T.Pm); E =  Mean total cross-sectional bone perimeter (B.Pm); F =  Mean polar moment of inertia (MMI). *<i>p</i><0.05, **<i>p</i><0.01 and *** <i>p</i><0.001 represents significant differences between strains, for each group. No statisticallly significant differences were found for each strain after receiving F.</p

Quantification of ALP activity in plasma from A/J and 129P3/J mice treated with 0, 10 or 50 ppm F in the drinking water for 8 weeks.

<p>Results are shown as mean ± SD of enzymatic activity (nmol of p-NP per min per mg of total protein). ^*Represents significant differences between strains for each group (<i>p</i><0.05). No statistical differences were found for each strain after receiving F.</p