Elevated vitamin D receptor levels in genetic hypercalciuric stone-forming rats are associated with downregulation of Snail

Abstract

Patients with idiopathic hypercalciuria (IH) and genetic hypercalciuric stone-forming (GHS) rats, an animal model of IH, are both characterized by normal serum Ca, hypercalciuria, Ca nephrolithiasis, reduced renal Ca reabsorption, and increased bone resorption. Serum 1,25-dihydroxyvitamin D [1,25(OH)(2)D] levels are elevated or normal in IH and are normal in GHS rats. In GHS rats, vitamin D receptor (VDR) protein levels are elevated in intestinal, kidney, and bone cells, and in IH, peripheral blood monocyte VDR levels are high. The high VDR is thought to amplify the target-tissue actions of normal circulating 1,25(OH)(2)D levels to increase Ca transport. The aim of this study was to elucidate the molecular mechanisms whereby Snail may contribute to the high VDR levels in GHS rats. In the study, Snail gene expression and protein levels were lower in GHS rat tissues and inversely correlated with VDR gene expression and protein levels in intestine and kidney cells. In human kidney and colon cell lines, ChIP assays revealed endogenous Snail binding close to specific E-box sequences within the human VDR promoter region, whereas only one E-box specifically bound Snail in the rat promoter. Snail binding to rat VDR promoter E-box regions was reduced in GHS compared with normal control intestine and was accompanied by hyperacetylation of histone H(3). These results provide evidence that elevated VDR in GHS rats likely occurs because of derepression resulting from reduced Snail binding to the VDR promoter and hyperacetylation of histone H(3).

Fig. 1

VDR is highly expressed in GHS rat intestine and kidney at the protein and mRNA levels. (A) Immunoblot for VDR and β-actin in total nuclear protein extracted from intestinal and kidney tissue from GHS and NC rats. Each lane represents intestinal or kidney extracts from an individual rat. All samples were run on the same blot. Lanes 1 to 4 are from NC rats; lanes 5 to 8 are from GHS rats. Lanes 1 and 5 are duodenum; lanes 2 and 6 are jejunum; lanes 3 and 7 are ileum, and lanes 4 and 8 are kidney. (B) Intestine and kidney VDR and GAPDH mRNAs from GHS and NC rats were subjected to real-time PCR using the primers in Table 1. VDR expression levels were normalized to GAPDH expression. VDR mRNA levels are increased in GHS versus NC rat duodenum, jejunum, and kidney. Results are individual values with mean shown as horizontal bars (n = 4 per group). *p = .027; **p = .034; ***p = .036.

Fig. 2

Snail is decreased in GHS rats at the protein and mRNA levels. (A, B) Immunoblots for Snail and β-actin of nuclear protein extracts from duodenum and kidney from individual GHS and NC rats. Representative results from an individual GHS and NC rat are shown. Snail protein levels were decreased in (A) duodenum and (B) kidney. (C) Total RNA isolated from intestinal and kidney tissue from GHS and NC rats was subjected to real-time PCR using primers listed in Table 1. Snail mRNA levels in GHS rat duodenum (*p = .026) and kidney (**p = .0002) were decreased (n = 4). Each data point represents intestinal or kidney extracts from a single rat. Snail levels in jejunum and ileum were not statistically significantly different between GHS and NC rats.

Fig. 3

Snail mRNA is inversely correlated with VDR mRNA. Total RNA isolated from GHS and NC rat intestine and kidney was subjected to real-time PCR using the primers listed in Table 1. Expression levels of VDR and Snail mRNA were normalized to GAPDH mRNA expression. Each data point represents both Snail and VDR measured on the same tissue sample from each of 31 rats. The Spearman rank correlation coefficient of the individual measurements reveals a significant inverse relationship (r = –0.507, p = .0031). The regression line y = 0.128 – 0.271x has an intercept of 0.055, and the SE of the slope is 0.095.

Fig. 4

Snail protein binds to E-box 3 within the rat VDR promoter. (A) Immunoblots of Snail and β-actin were performed 48 hours following transient transfection of HEK293 cells with vectors containing either the empty control (pcDNA3) or the Snail gene (pcDNA3-Snail). EMSA shows the binding of Snail to oligonucleotides containing sequences for E-box 1 (B), E-box 2 (C), and E-box 3 (D). Unlabeled oligonucleotides were used as competitors. In panels B, C, and D, lane 1 shows the binding of labeled E-box probe with pcDNA3-Snail nuclear extract; lane 2 shows unlabeled oligonucleotides competing with labeled oligonucleotide probes for binding to the DNA-protein complex; lane 3 shows DNA-protein complex formation in the presence of Snail antibody; and lane 4 shows lack of inhibition of the DNA-protein complex by normal IgG. Note that Snail does not specifically bind to either E-box 1 (B) or E-box 2 (C) sequences but does bind specifically to E-box 3 sequence (D).

Fig. 5

Snail binds to the VDR proximal promoter in vivo. (A) RNA extracted from HEK293, DLD1, and SW480 cells was subjected to real-time RT-PCR using specific primers for VDR, Snail, and β-actin. The relative expression levels of VDR and Snail in HEK293 were assigned a value of 1.0. Values are mean ± SEM for an n = 4 per experimental group. *p < .05; **p < .001. (B) Nuclear extracts from HEK293, DLD1, and SW480 cells were subjected to immunoblot using antibodies against VDR, Snail, and β-actin. β-actin was used as an internal control. (C) ChIP assays were performed on extracts of SW480, HEK293, and DLD1 cells using anti-Snail antibody. Normal IgG was used as a negative control. DNA was precipitated with either anti-Snail antibody or normal IgG. The primer pair used to amplify the human VDR promoter is described in “Materials and Methods.” Shown is a representative amplification of the input, DNA precipitated by the anti-Snail antibody, and normal IgG. The ChIP assay was repeated at least twice for each cell line.

Fig. 6

Hyperacetylation of histone H₃ (AcH₃) around the rat VDR promoter region in GHS rats. (A) ChIP assay on chromatin extracted from intestine of GHS and NC rats using anti-Snail antibody. The normal IgG was the negative control. Note the greater level of Snail detected in nuclear extracts of intestinal cells from NC rats. (B) AcH₃ was detected in chromatin extracts from intestine and kidney from GHS and NC rats by ChIP assays using anti-AcH₃ antibody. The PCR products of input and DNA when precipitated with anti-AcH₃ antibody or normal IgG using the same primers as in panel A revealed greater acetylation of histone H₃ in the GHS rat VDR promoter. The tissue ChIP assays were repeated at least twice for each sample of intestine and kidney tissue.

Fig. 7

Immunolocalization of intestinal VDR and Snail. VDR (A, D) and Snail (B, E) were coexpressed (C, F) in nuclei of small intestinal epithelial cells. VDR was expressed in a greater epithelial population and with higher signal intensity than Snail. Fluorescence staining of VDR and Snail revealed that both were coexpressed in the nuclei of cells composing the intestinal villi (A–C) and crypt glands (D–F).

Fig. 8

Immunolocalization by fluorescence staining of renal VDR and Snail. VDR (A, D) and Snail (B, E) were coexpressed (C, F) in renal epithelial cells of the loop of Henle (A–C) and the distal convoluted tubule (D–F). VDR was expressed in a greater epithelial population and with higher signal intensity than Snail.