Subacute ruminal acidosis is induced by high concentrations of short-chain fatty acids (SCFA, mainly acetate, propionate, and butyrate) that release protons to decrease the pH of the ruminal digesta. This low pH, in turn, is thought to damage epithelial barrier function. The present study applied a model of simulated ruminal acidosis ex vivo to investigate if SCFA directly contribute to epithelial barrier failure beyond their role as proton donors. Epithelial tissues from the rumen of slaughtered sheep were mounted in Ussing chambers and incubated under 3 different conditions. Two groups were incubated in the absence of SCFA at mucosal pH 6.1 (control) and pH 5.1, respectively, for 7 h. A third group was first incubated in a mucosal solution containing 100 mM SCFA at pH 5.1 for 2 h and, thereafter, in a mucosal solution without SCFA at pH 6.1 for the remaining 5 h. Transepithelial conductance (G), short-circuit current (I), and fluorescein fluxes were determined. After 7 h of incubation, the expression levels of claudin-1, claudin-4, claudin-7, and occludin were measured by quantitative reverse-transcription PCR and Western blot. Furthermore, the local distribution of these tight junction (TJ) proteins was examined by confocal laser scanning microscopy. A 7-h incubation at pH 5.1 in the absence of SCFA did not influence either G or fluorescein flux rates of ruminal tissues ex vivo compared with the control. In contrast, incubation at pH 5.1 with SCFA for only 2 h induced increases in G and fluorescein flux rates that continued even after tissues were returned back to pH 6.1. Expression analysis showed that pH 5.1 without SCFA for 7 h induced no changes in mRNA expression of claudin-1, claudin-4, claudin-7, and occludin and a selective decrease in protein expression of only claudin-4 compared with the control. However, a 2-h incubation at pH 5.1 in the presence of SCFA decreased the mRNA-expression of claudin-7, as well as the protein expression of claudin-4, claudin-7, and occludin. The decreased expression of these TJ proteins in the group incubated with SCFA was also evident in immunohistochemistry. Immunohistochemistry additionally evidenced a considerable retraction of all tested TJ proteins out of the TJ in that group. We conclude that a low mucosal pH of 5.1 is tolerated well by ruminal epithelia for several hours. However, a low pH in combination with SCFA induces damage to the TJ and disturbs barrier function, which is not immediately reversible upon the removal of the acidotic insult.

The objective of this study was to investigate whether individual short-chain fatty acids (SCFA) have a different potential to either regulate the formation of the ruminal epithelial barrier (REB) at physiological pH or to damage the REB at acidotic ruminal pH. Ruminal epithelia of sheep were incubated in Ussing chambers on their mucosal side in buffered solutions (pH 6.1 or 5.1) containing no SCFA (control), 30 mM of either acetate, propionate or butyrate, or 100 mM acetate. Epithelial conductance (Gt), short-circuit current (Isc), and fluorescein flux rates were measured over 7 h. Thereafter, mRNA and protein abundance, as well as localization of the tight junction proteins claudin (Cldn)-1, -4, -7, and occludin were analyzed. At pH 6.1, butyrate increased Gt and decreased Isc, with additional decreases in claudin-7 mRNA and protein abundance (each P < 0.05) and disappearance of Cldn-7 immunosignals from the stratum corneum. By contrast, the mRNA abundance of Cldn-1 and/or Cldn-4 were upregulated by 30 mM propionate, 30 mM butyrate, or 100 mM acetate (P < 0.05), however, without coordinated changes in protein abundance. At luminal pH 5.1, neither Gt, Isc, nor TJ protein abundance was altered in the absence of SCFA; only fluorescein flux rates were slightly increased (P < 0.05) and fluorescein signals were no longer restricted to the stratum corneum. The presence of acetate, propionate, or butyrate at pH 5.1 increased fluorescein flux rates and Gt, and decreased Isc (each P < 0.05). Protein abundance of Cldn-1 was decreased in all SCFA treatments but 30 mM butyrate; abundance of Cldn -4 and -7 was decreased in all SCFA treatments but 30 mM acetate; and abundance of occludin was decreased in all SCFA treatments but 30 mM propionate (each P < 0.05). Immunofluorescence staining of SCFA-treated tissues at pH 5.1 showed disappearance of Cldn-7, discontinuous pattern for Cldn-4 and blurring of occludin and Cldn-1 signals in tight junction complexes. The fluorescein dye appeared to freely diffuse into deeper cell layers. The strongest increase in Gt and consistent decreases in the abundance and immunosignals of tight junction proteins were observed with 100 mM acetate at pH 5.1. We conclude that SCFA may contribute differently to the REB formation at luminal pH 6.1 with possible detrimental effects of butyrate at 30 mM concentration. At luminal pH 5.1, all SCFA elicited REB damage with concentration appearing more critical than SCFA species.

Epithelial barrier dysfunction in LC occurs through downregulation of claudin-4, -5, and -8, and redistribution of claudin-5 and -8 off the tight junction, which contributes to diarrhea by a leak-flux mechanism. The key effector cytokines TNFα and IFNγ turned out to be the trigger for redistribution of claudin-5 and -8. Thus, alongside sodium malabsorption, leak-flux is yet another important diarrheal mechanism in LC.