Expression patterns of intestinal calcium transport factors and ex-vivo absorption of calcium in horses

Abstract

Background: In many species, the small intestine is the major site of calcium (Ca(2+)) absorption. The horse differs considerably from most other species with regard to the physiology of its Ca(2+) metabolism and digestion. Thus, this study was performed to get more information about the transcellular Ca(2+) absorption in the horse.Two mechanisms of intestinal Ca(2+) absorption are described: the passive paracellular pathway and the active, vitamin D-dependent transcellular pathway. The latter involves the following elements: vitamin D receptors (VDR), transient receptor potential vanilloid channel members 5 and 6 (TRPV5/6), calbindin-D9k (CB), the Na/Ca exchanger (NCX1) and the plasma membrane Ca-ATPase (PMCA). The aim of the present study was to investigate the protein and mRNA expression patterns of VDR, CB and TRPV6 and the ex-vivo Ca(2+) absorption in horses, assessed by qualitative and quantitative RT-PCR, western blot, immunohistochemistry and the Ussing chamber technique.

Results: Highest CB and TRPV6 mRNA levels were detected in the duodenum as compared to the middle parts of the jejunum and ileum and several sites of the large intestine. VDR mRNA levels did not change significantly throughout the intestine. TRPV5 mRNA was not detectable in the horse intestine. The highest VDR and CB protein levels were measured in the duodenum. Ussing chamber studies revealed ex-vivo Ca(2+) absorption only in the duodenum, but not in cecum and specific sites of the colon.

Conclusion: The present findings suggest that TRPV6, CB and VDR may be involved in active intestinal Ca(2+) absorption in horses, as described for other mammals. TRPV5 may not play a major role in this process. Furthermore, the expression patterns of these Ca(2+) transport elements and the results of the Ussing chamber procedure indicate that a significant part of active intestinal Ca(2+) absorption occurs in the duodenum in this species.

Figure 1

Reverse transcriptase PCR. Detection of VDR, calbindin-D9k (CB), TRPV6 and TRPV5 cDNA in different intestinal segments (DD = duodenum, JE = jejunum, IL = ileum, CC = cecum, CAV = colon ascendens ventrale, CAD = colon ascendens dorsale, CD = colon descendens) detected by reverse transcriptase PCR. GAPDH was used as a loading control. VDR, TRPV6 and CB cDNA was found in all investigated segments (Figure 1A, B, C), while TRPV5 was solely detected in the kidney (KI), not in the horse intestine (Figure 1D).

Figure 2

Real-time PCR. VDR, CB, and TRPV6 mRNA expression levels in the different intestinal segments in the horse intestine (DD = duodenum, JE = jejunum, IL = ileum, CC = cecum, CAV = colon ascendens ventrale, CAD = colon ascendens dorsale, CD = colon descendens) were determined by real-time (TaqMan) PCR. Expression was normalised to the expression of β-actin. VDR mRNA was found in each intestinal segment, but the amount and distribution patterns showed distinct inter-individual variations between the animals investigated. There were no significant differences between VDR mRNA expression levels in the small and large intestine (p > 0.05) (Figure 2A). The duodenum expressed the highest levels of TRPV6 and CB mRNAs (p < 0.05) (Figure 2B, C).

Figure 3

Western blot analyses. Expression of VDR and CB proteins in the horse intestine was determined by western blot analysis. Tissue samples of the duodenum (DD), jejunum (JE), ileum (IL), cecum (CC), colon ascendens ventrale (CAV), colon ascendens dorsale (CAD) and colon descendens (CD) were used. The loading was controlled by ß-actin. A) A representative western blot of VDR protein expression is shown. In all of the animals, major bands of approximately 40 kDa were found in the DD, while minor bands were detectable in the jejunum and colon in three animals. B) Relative expression of VDR to ß-actin was determined by densitometry using ImageJ software. The highest expression was measured in the DD. C) The strongest bands of CB protein were detected in the duodenum in all of the animals. Minor bands were visible in the jejunum. In 9 animals, weak signals were found in cecum, and in 6 animals, weak bands were seen in the ileum and colon ascendens ventrale. D) The expression of CB protein relative to ß-actin was determined by measuring the optical density using ImageJ software. The highest CB protein expression was found in the DD.

Figure 4

Immunohistochemistry VDR. Immunohistochemical analyses were performed to localise the VDR protein in the horse intestine. A) VDR protein was mainly detected in the superficial crypts of the duodenum. B) Tissue from Figure 4A at a higher magnification. Inside the enterocyte, the strongest labelling was found in the nuclei, while weaker signals were seen in the cytoplasm. C) Negative control. V = villus, C = crypt, S = submucosa.

Figure 5

Immunohistochemistry Calbindin-D9k. Immunohistochemistry was performed to localise calbindin-D9k expression in the intestinal wall. The strongest labelling was visible in the villi of the duodenum (DD) followed by that of the jejunum (JE). Considerably weaker staining was observed in the cecum (CC) and in the colon ascendens ventrale (CAV). Inside the enterocytes, the strongest staining was seen in the cytoplasm.

Figure 6

Ussing chamber. Ex-vivo Ca²⁺ absorption in different intestinal segments (duodenum = DD, cecum = CC, colon descendens dorsale = CAD, colon descendens = CD) was measured by the Ussing chamber technique. Positive flux rates (absorption) from the mucosal to the serosal sides were measured in the duodenum, while in the remaining segments (CC, CAD, CD), the negative flux rates (secretion) were determined.