Determination of mRNA Expression of Typical Proteins of Tight Junctions in the Intestinal Mucosa of Broilers ( Gallus gallus domesticus )

Tight junctions are mainly formed by two types of proteins; claudins and occludin, both of which are fundamental to maintain the integrity and barrier function of the intestinal epithelium. This barrier function allows for the absorption of nutrients, mainly by transcytosis; however, in birds, 90% of the substances are absorbed by paracellular mechanisms. Despite this, claudins present in the different parts of the intestinal tract of adult chickens are not known, much less their functional role. This study aimed to determine the presence of mRNA of claudins 1, 2, 3, 5, 10, 12, 16 and occludin, in the different regions of the intestine (duodenum, jejunum, ileum, cecum, and rectum) in chickens (Gallus gallus domesticus) through RT-PCR. To meet this goal, 7 weeks old roosters destined for slaughter and chicken embryos of 16 days of incubation (positive control) were used. For all the processed samples, amplicons of the expected size were obtained; claudin 1 (662 pb), claudin 2 (162 pb), claudin 3 (185 pb), claudin 5 (224 pb), claudin 10 (687 pb), claudin 12 (738 pb), claudin 16 (191 pb) and occludin (430 pb). To corroborate these findings, obtained amplicons were sequenced and, subsequently, a basic alignment was performed on the NCBI, obtaining a correlation of 100% with the original sequences in all analyzed samples. To our knowledge, the present work represents the first written report regarding the presence of mRNA of the main proteins involved in tight junction formation throughout the intestinal tract of domestic chickens of 7 weeks of age. These findings will allow elucidating the specific function of each of the reported proteins in the process of paracellular absorption in chickens.


Introduction
Tight junctions are an element of the junction complex found in epithelial and endothelial cells, they form a belt between the plasmatic membrane lateral domain near the apical pole, which serves as a primary barrier to the diffusion of solutes, regulates paracellular transit, and enables cell polarization. Tight junctions are fundamental to maintain the integrity and paracellular barrier function of epithelium and endothelium cells [1] [2]. They restrict the movement of proteins between the basolateral and apical compartments, regulating the passage of ions and solutes through the paracellular space [1] [3].
Structurally, tight junctions are composed of proteinaceous filament nets, called strands which traverse the extracellular space to interact with the tight junctions of adjacent cells. The physiological property to act as a seal depends on several factors such as the number of strands, the amount of physical tension exerted on the seal, and the primary component of strands' proteins [4]. Tight junctions have been studied mainly in humans and mice, in these species, it was determined that the differentiation of claudins is evident along the length of the small intestine, where these proteins are vital for the appropriate absorption of nutrients [5].
Claudins are transmembrane proteins with a molecular weight ranging from 20 to 27 kDa, they polymerize linearly to form strands, establishing transcellular interactions in cis (between claudins of the same cell) and in trans (between claudins of adjacent cells), which determines paracellular permeability, epithelial polarization, and conservation of transepithelial resistance, as well as the selective passage of molecules and ions [1] [2].
This multigenic protein family consists of approximately 27 members in humans and mice [2] [4] [12] [13] [14], species in which most of these proteins have been studied, although their presence has also been observed in different organs in chicken embryos and adult birds [3] [15] [16] [17].
Occludin is a highly conserved protein in mammals and birds [25]. It is present in cells that form tight junctions. The size of the occludin varies with the species; for example, in chickens, it is 55.9 -62 kDa, while in human is 82 kDa [12] [26] [27] [28]. This protein is not essential for the assembly of a tight junction, but it is important when limiting functional permeability [6].
The intestinal absorption occurs by the passage of molecules through the cell and by paracellular transit [6] [11] [29]. The paracellular transit works by one of the following mechanisms: the "pore" pathway, which is of low capacity and allows the passage of ions and small solutes.  [36].
The digestive tract of the bird is shorter and simpler than that of mammals; however, it is highly efficient for digestion and nutrient absorption processes [37]. In the intestinal epithelium of birds, 90% of the substances (ions, nutrients, etc.) are absorbed through pores 4 to 8 nm in diameter, which is present in the tight junctions, this could explain why birds have a greater absorption capacity of water-soluble metabolites compared to mammals [8] [38]. Additionally, it has been reported that alterations in the expression of tight junctions' proteins cause alterations in the function of claudin-based paracellular barrier and are related to water imbalance, inflammation, cancer, and brain disease [1] [18].
Tight junctions play a preponderant role during intestinal absorption in birds.
For this reason, it is essential to identify the claudins by the determination of the presence of claudin mRNA in the mucous tunic of the different regions of the intestine in chickens 7 weeks of age, which we consider a fundamental step on the way to elucidate their specific role.  (Table   1 and Table 2) and to evaluate its integrity, samples were subjected to electrophoresis in 1.5% agarose gels stained with ethidium bromide and observed on the transilluminator [40] (data not shown).

RT-PCR
For each of the genes, the synthesis of cDNA with reverse transcriptase of M-MLV was performed (Invitrogen, Cat. 28025013) and the quantity and purity of the cDNA were estimated. The presence of the different claudins was determined

Mucosa
In adult chickens, the mucous tunic of the different intestinal segments was harvested using the technique of scraping with scalpel blade, and verification by routine histological processing and stained with H-E was performed. As can be seen, only the components of the mucous tunic and no other structural component are appreciated ( Figure 1).

Characteristics of purified RNA
All RNAs obtained were evaluated by spectrophotometry. In adult chickens, the concentrations obtained were between 70.9 and 1398.7 ng/ml, while in embryos they were between 1137.9 and 2002.4 ng/ml. Consistently, a greater concentration of RNA was obtained from the embryos than in adult chickens because in the embryos we take the four structural tunics of the intestinal portions, while in adult chickens only the mucous tunic. To know the degree of purity, the ratio of the readings 260/280 nm was determined, the values obtained were in the range of 2.0 -2.5, which shows high purity of isolated RNA (Table 1 and Table 2). The values obtained allow us to consider that the purification method was adequate. Finally, all samples were subjected to electrophoresis in agarose gels and in all of them, the bands corresponding to ribosomal RNA were observed (data not shown).

Amplification of the fragments by RT-PCR
The products expected for each of the genes under study were obtained by end-point RT-PCR, both for the positive control (chicken embryos) and the  (Figure 2).

Blast alignment
The reported sequences had the necessary characteristics to perform the basic alignment in the Blast program, 100% of identity, concerning the expected gene for all samples was found, so we can say that the obtained amplicons correspond to the indicated genes.

Discussion
Karcher et al. (2008) [8] report that 90% of the nutrients are absorbed by paracellular transit in the epithelium of the small intestine in birds. In a previous job, we determined that the avian intestinal tract has villi throughout its length [37], suggesting that the cecum and rectum also participate in the absorption process.
For this reason, we considered necessary to include these organs in the present study. In this work, we determined the presence of the mRNAs of the proteins studied in all portions of the intestine, both in chicken embryos and 7-week-old   [44] and ileum and cecum of chickens [16].
The latter is consistent with our results, although we also found it to be present in duodenum, jejunum, and rectum.
Claudin 5 is expressed predominantly in the endothelium and has been reported in the intestinal epithelium of adult rats and mice [19] [20] [43]; as well as in different periods of embryonic development, in the small intestine epithelium in chickens [39], and the blood-brain barrier [45]. In this study, the pres-   [19], reported that in mice it is expressed throughout the gastrointestinal tract, where it was found at the bottom of the crypts. Its presence has also been reported in the duodenum and jejunum of laying hens, where it participates in the absorption of calcium by paracellular route [17]. We found it in all intestinal segments of the birds under study. Claudin 10 is expressed in renal tubules, kidney medulla, and intestines [42]. In the present study, the presence of mRNA for claudin 10, in the duodenum, jejunum, and ileum of embryos was observed, which coincides with that reported by The study of the proteins that form the tight junctions will allow us to better understand the mechanism that regulate their modification in the different physiological and pathological states of the intestine, particularly in the absorption processes where birds preferentially use the paracellular pathway. It will also al- On the other hand, we will understand how the close junctions are affected by specific pathogens of the intestinal tract, either by direct interaction with intestinal cells or by their toxins. It would also allow the generation of new therapeutic strategies against this type of disease [53]- [59].

Conclusion
This work is the first report about the mRNA expression of proteins involved in the formation of tight junctions (occludin and claudins 1, 2, 3, 5, 10, 12, 16) in the mucous tunic of all portions of the small and large intestine (duodenum, jejunum, ileum, cecum, and rectus) in the chicken (Gallus gallus domesticus).
These findings will allow us to further elucidate the specific function of each of the studied proteins in the process of paracellular absorption in adult chickens.