Effect on colonic calcium transporters, phosphatases and claudin

In line with our previous study^{(Reference Hu, Hendriks and van Baal2)}, post-ileal Ca absorption in pigs was also observed by others (e.g. Liu et al. ^{(Reference Liu, Bollinger and Ledoux17)}), but the molecular mechanisms clarifying this observation remained to be elucidated. Here, we demonstrate that phytase inclusion tended to elevate the expression of CLDN-12 mRNA in the colonic mucosa of pigs. A previous study in mice and Caco-2 cell line demonstrated that CLDN-2 and CLDN-12 form pores permitting Ca ions to pass through, and their expression was upregulated by 1,25(OH)₂D₃ exposure^{(Reference Fujita, Sugimoto and Inatomi18)}. This indicates that the enhanced expression of CLDN-12 in response to phytase inclusion might improve the colonic mucosal permeability to Ca.

Contrary to CLDN-12, expression of TRPV5, TRPV6 and CaBP-D9k was reduced with phytase and high Ca intake. Expression of Ca transporters in the colon of pigs has not been reported previously. In vivo studies in mice demonstrated that a high dietary Ca level reduced mRNA levels of TRPV6 in the proximal colon^{(Reference Lee, Liu and O’Neill19)}. Using the Ussing chamber technique, the researchers in the same study further demonstrated that the application of high extracellular Ca concentration inhibited transcellular Ca absorption in proximal colon tissues of mice. Their results indicate that colonic Ca absorption, particularly active transcellular Ca absorption, is reduced by a high dietary Ca intake, which was in line with our observations in pigs. This modulation of Ca transport might be mediated via the action of PTH and vitamin D₃ ^{(Reference Sassi, Tamone and D’Amelio20)}, as we observed that the inhibition of expression of TRPV5, TRPV6 and CaBP-D9k in response to high dietary Ca intake was accompanied by a lower serum PTH content. We propose that the elevated serum Ca content in response to high dietary Ca intake inhibits PTH secretion by stimulating CaSR expressed in the parathyroid gland^{(Reference Chen and Goodman21)}. A reduction of PTH, in turn, decreases circulatory 1,25(OH)₂D₃ to limit renal Ca reabsorption by the upregulation of renal expression of CYP24 mRNA, encoding a mitochondrial monooxygenase responsible for the modification and inactivation of bioactive 1,25(OH)₂D₃. The observed downregulation of TRPV5 and TRPV6 mRNA expression in the colon could be ascribed to the reduced concentration of serum 1,25(OH)₂D₃ since the genes of these transporters contain a vitamin D₃ receptor -responsive element in their promoter region^{(Reference Hoenderop, Nilius and Bindels22)}. The upregulated expression of vitamin D₃ receptor in both jejunal and colonic mucosa in response of high Ca and/or phytase intake supports this idea. In mice, Ca-sensing by CaSR located in the intestine may decrease the expression of TRPV6 and CaBP-D9k directly^{(Reference Lee, Liu and O’Neill19)}. However, this mechanism seems to be unlikely in pigs since CaSR mRNA was beyond the limit of detection in both jejunal and colonic mucosa in the present study (data not shown).

We determined the mRNA expression of mucosal phosphatases (MINPP1 and IAP) and inositol transporters (SMIT) assuming that their expression level would be affected by our dietary treatments. However, IAP mRNA was beyond the limit of detection in colon and not affected by dietary treatments in jejunum. Moreover, MINPP1 mRNA was not altered by phytase inclusion or dietary Ca intake in both intestinal segments. The MINPP1 is an intercellular phosphatase located in endoplasmic reticulum. Recent in vitro studies in the Caco-2^{(Reference Briviba, Schollenberger and Rodehutscord23)} and H1299 cell line^{(Reference Windhorst, Lin and Blechner24)} indicate that MINPP1 is secreted into the medium and can catalyse dephosphorylation of extracellular IP. It is, therefore, possible that MINPP1 is involved in the degradation of IP in the intestine. Previous studies in broilers indicated that high Ca intake reduced mucosal phosphatase efficacy to degrade IP in the brush-border vesicles of the small intestine^{(Reference Applegate, Angel and Classen25)}. Brun et al. ^{(Reference Brun, Traverso and Rigalli26,Reference Brun, Brance and Rigalli27)} also reported that high Ca intake modified the activity of IAP without affecting its protein expression level in the small intestine of rats. These studies support our results and indicate that dietary Ca intake or phytase does not affect the level of IAP expression to regulate the efficacy of IAP. Our results also demonstrated that colonic expression of SMIT was significantly reduced with high dietary Ca intake although this effect was minor. We observed that high Ca intake reduced the degradation of IP in the small intestine in pigs^{(Reference Hu, Hendriks and van Baal2)}, which subsequently promotes the availability of IP for degradation into inositol in the colon.

The colon plays an important role in mineral and water absorption in pigs^{(Reference Breves, Kock and Schröder28)}. The reduction of mRNA expression of CLDN-4, CLDN-7 and CLDN-10 in the colon indicates that high Ca intake impaired paracellular permeability for anions and cations in the porcine colon. Unfortunately, the ion permeability of these CLDN members is still debated. Van Itallie et al. ^{(Reference Van Itallie, Rahner and Anderson11)} reported that CLDN-4 could mediate Cl permeation, while Hou et al. ^{(Reference Hou, Renigunta and Yang29)} argued that it builds pores for Na. Experiments to unravel the ion selectivity of CLDN-7 led to controversial results; the protein may mediate transepithelial permeation of Mg^{(Reference Thongon and Krishnamra30)} or Cl^{(Reference Fan, Tatum and Hogard31)}. Other studies (e.g. Van Itallie et al. ^{(Reference Van Itallie, Rogan and Yu32)}) demonstrated the existence of two splice variants of CLDN-10, designated CLDN-10a and CLDN-10b, each with different physiological functions in mice. By contrast, Gunzel et al. ^{(Reference Gunzel, Stuiver and Kausalya33)} reported the existence of six different isoforms of CLDN-10 as a result of alternative splicing in mice and humans. We performed an intensive Blast search (Build Sscrofa11.1) and analysed two porcine CLDN-10 homologues: CLDN-10a (accession no. XM_021065094) and CLDN-10b (accession no. XM_021065095). In the present study, we used a primer set that hybridises to a completely identical region, shared by both isoforms in order to determine the sum expression level. Taken together, downregulation of CLDN-4, CLDN-7 and CLDN-10 may alter the mucosal paracellular permeability to traverse certain minerals across the colonic epithelial layer. It remains unknown which specific minerals are involved.

Effect on jejunal calcium transporters and claudin

Similar to the colon, high Ca and phytase intake reduced TRPV6 expression in jejunal mucosa. Gonzalez-Vega et al. ^{(Reference Gonzalez-Vega, Liu and McCann34)} reported that incremental dietary Ca intake linearly reduced jejunal expression of TRPV6 and CaBP-D9k, which is in agreement with our results. Noteworthy, in our study jejunal TRPV5 expression was not affected by dietary Ca level, whereas it was not reported by Gonzalez-Vega et al. ^{(Reference Gonzalez-Vega, Liu and McCann34)}. An explanation may be that TRPV5 in mice is primarily expressed in the kidney, while TRPV6 is ubiquitously expressed^{(Reference Khanal and Nemere3)}. Indeed, we found that TRPV6 expression was approximately 30 and 50 times higher than TRPV5 in porcine jejunal and colonic mucosa, respectively. By contrast, renal expression of TRPV5 in pigs was approximately 6 times higher than TRPV6 (Hu et al. unpublished). Collectively, it appears that TRPV5 is less involved in intestinal Ca absorption compared with TRPV6. We also observed an upregulation of TRPM7 with dietary Ca intake in the jejunal mucosa. However, unlike TRPV5 and TRPV6, two channels highly selective for Ca^{(Reference den Dekker, Hoenderop and Nilius35)}, TRPM7 seems to be less selective with higher affinity for Zn, Mg and Mn than Ca^{(Reference Bates-Withers, Sah and Clapham36)}. An upregulation of TRPM7 might be indicative of an antagonistic impact of a high dietary Ca against other cations.

Contrary to TRPV5 and TRPV6, CLDN-2 and CLDN-12 mRNA expression in the jejunal mucosa was enhanced by high Ca and phytase intake, respectively. This regulation pattern of CLDN-2 and CLDN-12 was in line with the soluble inorganic Ca content in the distal small intestine, which indicated that Ca was transported in the direction of the electrochemical gradient across the epithelial layer with high Ca intake but against it with low Ca intake (online Supplementary Table 1). It could be that low dietary Ca intake reduced CLDN-2 expression in order to prevent the backflow of Ca to the luminal side. By contrast, high dietary Ca intake enhanced CLDN-2 expression probably to increase the capacity of passive Ca uptake as an energy-saving process. Taken together, the inhibitory action on the expression of TRPV6, and also TRPV5 and CaBP-D9k in the colon, and stimulatory effect on Ca permeable CLDN-2 and CLDN-12 with high Ca intake suggest that paracellular Ca permeation was minor at a low but more pronounced with high Ca intake. Based on these findings, it is postulated that Ca absorption shifts from transcellular to paracellular pathway with high dietary Ca and phytase intake.

Compared with CaBP-D9k, transcript levels of CaBP-D28k were very low in the jejunal mucosa, even absent in the colonic mucosa. This indicates that CaBP-D9k may play a greater role in shuttling Ca across cytoplasm in the GIT of pigs. Christakos et al. ^{(Reference Christakos, Mady, Dhawan, Feldman, Pike and Bouillon37)} reviewed that CaBP-D28k was expressed highest in the avian intestine and mammalian kidney, while CaBP-D9k was observed only in mammals and was abundantly expressed in the mammalian intestine. Indeed, we observed a much higher expression of CaBP-D28k compared with CaBP-D9k in the kidney of pigs (Hu et al. unpublished). Thus, the distribution of CaBP-D28k and CaBP-D9k appears tissue-specific in pigs. Moreover, despite the similar names, the physiological role of CaBP-D28k may differ from CaBP-D9k. A previous in vivo study using transgenic mice^{(Reference Zheng, Xie and Li38)} indicated that mice lacking CaBP-D28k but with normal expression of CaBP-D9k developed little calcaemic abnormalities; by contrast, mice expressing only 10 % of CaBP-D9k but with normal expression level of CaBP-D28k developed hypocalcaemia and rickets. It seems that the Ca buffering function of CaBP-D28k can be largely compensated by CaBP-D9k, but not vice versa. It is, therefore, possible that CaBP-D9k plays a greater role in the transit of Ca across the intestinal mucosa to serve whole body Ca homeostasis.

Effect on phosphorous transporters

Instead of NaPi-IIb, we detected NaPi-IIc in the porcine jejunum. Moreover, all type II transporters were absent in the colonic mucosa. Apparently, NaPi-IIc is the most important Na-P cotransporter in the small intestine of pigs. Wubuli et al. ^{(Reference Wubuli, Reyer and Murani39)} investigated the tissue-wide mRNA expression status of all currently annotated P transporters. Their results combined with our observation indicate that NaPi-IIc is the only type II P transporter abundantly expressed in the GIT of pigs and that this isotype contributes to intestinal P absorption. We demonstrated that high Ca uptake reduced intestinal P absorption in the small intestine of these pigs^{(Reference Hu, Hendriks and van Baal2)}. Here, we found that the level of NaPi-IIc mRNA was neither affected by dietary Ca level nor phytase inclusion, suggesting that the reduction of P absorption was attributed to a lower NaPi-IIc activity which was regulated at the posttranscription level, e.g. vesicle trafficking of NaPi-IIc to-and-from the apical membrane.

We observed a nominally (P = 0·055) elevated expression of PiT-1 mRNA in the colon when pigs were fed a high Ca compared with low Ca diet. Previous transgenic studies showed that overexpression of PiT-1 enhanced serum P and reduced serum Ca in both rats^{(Reference Suzuki, Ammann and Nishiwaki-Yasuda40)} and mice^{(Reference Chande, Ho and Fetene41)}. These data raise the possibility that high dietary Ca led to insoluble Ca–P complexes impairing intestinal P absorption and serum P content. A higher expression of PiT-1 may increase the capacity of P absorption in the pig’s colon to support systemic P homeostasis.

Paracellular P permeation might also substantially contribute to intestinal P absorption in pigs. Soluble inorganic P content was much lower in the serum than in the digesta in all GIT segments (online Supplementary Table 1), which generated a wide electrochemical gradient difference of P across the epithelium cell. To the knowledge of the authors, paracellular P absorption in pigs has not been reported before. Using the in situ ligated intestinal loop technique, Marks et al. ^{(Reference Marks, Lee and Nadaraja42)} demonstrated in rats that only 30 % of the intestinal P absorption was Na dependent, irrespective of P content in the buffer solution instilled into the GIT segments. Their results suggest that P absorption might be primarily mediated via paracellular permeation in the GIT. This assumption is in line with an in vivo study in pigs^{(Reference Stein, Kadzere and Kim43)} which demonstrated that incremental dietary P level linearly enhanced total tract P digestibility. Thus, P absorption is highly dependent on luminal P content, which suggests that paracellular P absorption may be substantial in pigs. However, the mechanism for P permeation remains unknown.

In conclusion, our data suggested that Ca absorption shifts from transcellular to paracellular pathways when dietary Ca is increased from deficient to abundant and also when Ca availability is enhanced with microbial phytase supplementation, as evidenced by downregulation of jejunal and colonic genes related to transcellular Ca absorption and upregulation of Ca pore-forming claudins (CLDN-2 and CLDN-12). Paracellular P absorption may be substantial in pigs as the expression of genes related to transcellular P absorption remain mostly unaffected by dietary Ca and phytase content.