The current study was performed to examine the expression patterns of the Ca2+ transport elements VDR, CB and TRPV6 in the horse intestine and further investigate the ex-vivo Ca2+ absorption via the Ussing chamber technique.
It is well documented that the small intestine is the main site of active intestinal Ca2+ absorption in many species [4, 15, 18]; however, marked variations between different species have been observed [17, 30–32].
With regard to the Ca2+ metabolism, horses show differences compared with most monogastric animals because they have remarkably high serum Ca2+ concentrations, low mean serum calcidiol and vitamin D concentrations, high intestinal Ca2+ absorption; they depend on the Ca2+ content in their feed, and excess Ca2+ is eliminated via the urine [18, 19, 24, 25, 33].
These physiological characteristics are similar to those of other hindgut fermenters such as rabbits and other small wild herbivores. Because the rabbit, a cecum fermenter, absorbs a significant amount of Ca2+ in the cecum [22, 23, 26], the question arises if the horse, as a hindgut fermenter, also shows peculiarities concerning the site of active intestinal Ca2+ absorption.
Therefore, the intestinal protein and mRNA expression of the Ca2+ transport elements VDR, TRPV6, and CB were investigated, and the active Ca2+ absorption was measured ex-vivo in different intestinal segments by the Ussing chamber technique.
High mRNA levels of TRPV6 and CB as well as high protein levels of VDR and CB in the duodenum indicated that this is the main site of Ca2+ absorption in the horse. These observations were further supported by the results of the Ussing chamber technique, demonstrating that the only measurable directed ex vivo Ca2+ transport through intestinal epithelium i.e. from mucosal to serosal side was found in the duodenum. These findings confirmed the idea that transcellular movements occur mainly in segments where the sojourn time of the chyme is very short so that a maximum amount of Ca2+ could be absorbed during this short period . Similar results have been documented in a Ussing chamber study performed by Cehak et al. , where highest ex-vivo Ca2+ absorption were measured in the duodenum of horses, followed in descending order by the jejunum, colon ascendens dorsale and cecum. In the present study, the duodenum was the only segment where the absorbance was measurable, while secretion of Ca2+ was recorded in the remaining investigated segments. The extent of active Ca2+ absorption is closely related to feeding [33, 36], and thus, the different measurements in the hindgut may be explained through the different Ca2+ content in the feed, particularly as reflected in our results. In addition to the VDR protein expression in the duodenum, protein bands were detectable in the hindgut in three animals. This result may indicate that active Ca2+ absorption occurs in the hindgut, and this absorption may be activated if dietary Ca2+ content is reduced or demand increased. This idea is supported by the detection of CB and TRPV6 mRNA in the hindgut, although the expression levels were low. The regulation of VDR has been reported in the hindgut of ruminants. Goff et al.  observed that VDR expression in Jersey cows was 3-4 fold higher during late pregnancy than in non-lactating cows. A significant decrease of VDR expression in the colon is described post-partum in sheep and goats by Liesegang et al. . Therefore, it is conceivable that the inter-individual variations between the animals used for this study are due to different physiological status or previous keeping and feeding of the animals. The information about the horses used for this study was limited to age and sex, so further studies with special feeding groups are recommended.
The localisation of VDR in the nuclei, particularly in the crypt, is discussed and observed in goats, cows and rabbits [23, 26, 39]. Compared with the expression of CB, which is primarily in the adult enterocytes, VDR is mainly expressed in younger cells.
Recently, Rourke et al.  measured the mRNA levels of TRPV5/6, CB, VDR, PMCA1 and NCX1 in the horse intestine. In contrast to our study, Rourke et al.  found higher levels of TRPV6 and CB mRNA in the proximal jejunum than in the duodenum. This divergence may be the result of different sampling because the samples for this study were taken from the middle part of the jejunum. Thus, the conclusion is restricted to the parts of the intestine investigated in this study, and further research on Ca2+ absorption and protein expression in the proximal jejunum are recommended.
Interestingly, a divergence in the expression of VDR protein and mRNA was observed in this study. The missing correlation resembles the phenomenon described by Mohri et al. , which found that human VDR is post-transcriptionally regulated by microRNAs that suppress the translation or degradation of VDR mRNAs. Based on the low VDR mRNA expression in the small intestine, Rourke et al.  argued that the transcellular epithelial Ca2+ transport in the horse is not as dependent on VD as in other species. The high levels of VDR proteins in the duodenum in the horses in this study suggested that this interpretation is not accurate and that posttranscriptional regulation of VDR mRNA may play an important role for intestinal Ca2+ absorption in the horse. Thus, VD may be involved in regulatory processes of active Ca2+ absorption in the horse intestine through VDR.
In contrast to Rourke et al. , which detected a low level of TRPV5 mRNA in the intestinal tract, we observed no signal in the samples in the present investigation, possibly because the amount of TRPV5 mRNA was under the detection limit.
As described for other mammals, TRPV5 may be the major isoform found in the kidney, whereas TRPV6 expression is primarily detectable in the intestine .