Volume 65, Issue 2, Pages (February 2004)

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Volume 65, Issue 2, Pages 531-539 (February 2004) Regulation of gene expression by dietary Ca2+ in kidneys of 25-hydroxyvitamin D3-1α- hydroxylase knockout mice Joost G.J. Hoenderop, Helena Chon, Dimitra Gkika, Hans A.R. Bluyssen, Frank C.P. Holstege, Rene St-Arnaud, Branko Braam, Rene J.M. Bindels Kidney International Volume 65, Issue 2, Pages 531-539 (February 2004) DOI: 10.1111/j.1523-1755.2004.00402.x Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 1 The effect high dietary Ca2+ intake on the serum Ca2+ concentration in 1α-hydroxylase (1α-OHase-/-) mice. Values are presented as means ± SEM (N = 6). *P < 0.05, significant different from 1α-OHase+/+ and 1α-OHase+/- mice fed a normal Ca2+ diet containing 1.1% (wt/vol) Ca2+; #P < 0.05, significant different from 1α-OHase-/- mice on normal Ca2+ diet containing 1.1% (wt/wt) Ca2+. Kidney International 2004 65, 531-539DOI: (10.1111/j.1523-1755.2004.00402.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 2 Number of genes regulated when comparing the three conditions 1α-hydroxylase (1α-OHase-/-) mice versus 1α-OHase+/- mice (left), 1α-OHase-/- mice versus 1α-OHase-/- mice supplemented with dietary Ca2+ (middle) and 1α-OHase+/- mice versus Ca2+ supplemented 1α-OHase+/- mice (right). The number of total-regulated (A), up-regulated (B), or down-regulated (C) transcripts on the DNA chip are depicted. Genes are shown in the overlapping regions that are regulated in two conditions. Kidney International 2004 65, 531-539DOI: (10.1111/j.1523-1755.2004.00402.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 3 Hierarchical clustering of all three comparisons [1α-hydroxylase (1α-OHase-/-) mice versus 1α-OHase+/- mice, 1α-OHase-/- mice versus 1α-OHase-/- mice treated with Ca2+ and 1α-OHase+/- mice compared to 1α-OHase+/- mice fed the enriched Ca2+ diet]. Genes that are significantly up- (dark) or down-regulated (light) are shown. Kidney International 2004 65, 531-539DOI: (10.1111/j.1523-1755.2004.00402.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 4 Differential expression patterns depicted in an intensity-dependent fashion using Cy3/Cy5 scatter plots. Regulated genes by inactivation of the 1α-hydroxylase (1α-Ohase) gene (A) and supplementation of dietary Ca2+ in the vitamin D–deficient state (B) form two distinct populations of genes. Genes that are regulated in two conditions (when 1α-OHase+/- mice are compared with 1α-OHase-/- mice and when 1α-OHase-/- supplemented with dietary Ca2+) are shown above the 0.7 log2 line (up-regulated in both comparisons) and below the -0.7 log2 line (down-regulated in both comparisons). Genes that are regulated in opposite directions in both comparisons are shown as large black dots. Kidney International 2004 65, 531-539DOI: (10.1111/j.1523-1755.2004.00402.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 5 A subset of regulated functional gene groups based on Gene Ontology (GO) with differential regulation due to 1α-hydroxylase (1α-OHase) inactivation or Ca2+ supplementation. Displayed GO groups contained at least five genes, had either a mean overall ratio of at least 0.3 or had at least 20% of the genes changed significantly in both comparisons. Box plots indicate the quartiles of the ratios. Kidney International 2004 65, 531-539DOI: (10.1111/j.1523-1755.2004.00402.x) Copyright © 2004 International Society of Nephrology Terms and Conditions