Background & Aims Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis. Methods We studied 3 mouse models of IBD: colitis induced by trinitrobenzene sulfonic acid, colitis induced by microflora (in gnotobiotic interleukin-10−/−), and colitis induced by adoptive transfer of CD4⁺CD45RB^high T cells. Effects of the tumor necrosis factor (TNF) and interferon (IFN)-γ on Kl expression and the activity of its promoter were examined in renal epithelial cells (mpkDCT4 and mIMCD3). Results Renal expression of Kl messenger RNA (mRNA) and protein was reduced in all 3 models of IBD. Reduced level of KL correlated with the severity of colitis; the effect was reversed by neutralizing antibodies against TNF. In vitro, TNF inhibited Kl expression, an effect potentiated by IFN-γ. The combination of TNF and IFN-γ increased expression of inducible nitric oxide synthase (iNOS) and increased NO production. The effect of IFN-γ was reproduced by exposure to an NO donor and reversed by the iNOS inhibitor. In cells incubated with TNF and/or IFN-γ, Kl mRNA stability was unaffected, whereas Kl promoter activity was reduced, indicating that these cytokines regulate Kl at the transcriptional level. Conclusions The down-regulation of KL that occurs during inflammation might account for the extraintestinal complications such as abnormalities in bone homeostasis that occur in patients with IBD.

Na⁺/H⁺exchanger 3 (NHE3) provides a major route for intestinal Na⁺absorption. NHE3 has been considered a target of proinflammatory cytokines and enteropathogenic bacteria, and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea. However, the possibility of loss of NHE3 function reciprocally affecting gut immune homeostasis has not been investigated. In this report, we describe that NHE3-deficient mice spontaneously develop colitis restricted to distal colonic mucosa. NHE3^−/−mice housed in a conventional facility exhibited phenotypic features such as mild diarrhea, occasional rectal prolapse, and reduced body weight. Genomewide microarray analysis identified not only a large group of transport genes that potentially represent an adaptive response, but also a considerable number of genes consistent with an inflammatory response. Histological examination demonstrated changes in the distal colon consistent with active inflammation, including crypt hyperplasia with an increased number of 5-bromo-2′-deoxyuridine-positive cells, diffuse neutrophilic infiltrate with concomitant 15-fold increase in matrix metalloproteinase 8 expression, an increased number of pSer²⁷⁶-RelA-positive cells, and a significant decrease in periodic acid-Schiff-positive goblet cells. Real-time PCR demonstrated elevated expression of inducible nitric oxide synthase (38-fold), TNF-α (6-fold), macrophage inflammatory protein-2 (48-fold), and IL-18 (3-fold) in the distal colon of NHE3^−/−mice. NHE3^−/−mice showed enhanced bacterial adhesion and translocation in the distal colon. Colitis was ameliorated by oral administration of broad-spectrum antibiotics. In conclusion, NHE3 deficiency leads to an exacerbated innate immune response, an observation suggesting a potentially novel role of NHE3 as a modifier gene, which when downregulated during infectious or chronic colitis may modulate the extent and severity of colonic inflammation.

Background & Aims NHE3 is a target of inhibition by proinflammatory cytokines and pathogenic bacteria, an event contributing to diarrhea in infectious and idiopathic colitis. In mice, NHE3 deficiency leads to mild diarrhea, increased intestinal expression of interferon (IFN)-γ, and distal colitis, suggesting its role in epithelial barrier homeostasis. Our aim was to investigate the role of NHE3 in maintaining mucosal integrity. Methods Control or dextran sulfate sodium (DSS)-treated, 6- to 8-week-old wild-type (WT) and NHE3^−/− mice were used for the experiments. Small intestines were dissected for further analysis. Results NHE3^−/− mice have elevated numbers of CD8α⁺ T and natural killer cells in the intraepithelial lymphocytes and lamina propria lymphocytes compartments, representing the source of IFN-γ. NHE3^−/− mice display alterations in epithelial gene and protein expression patterns that predispose them to a high susceptibility to DSS, with accelerated mortality resulting from intestinal bleeding, hypovolemic shock, and sepsis, even at a very low DSS concentration. Microarray analysis and intestinal hemorrhage indicate that NHE3 deficiency predisposes mice to DSS-induced small intestinal injury, a segment never reported as affected by DSS, and demonstrate major differences in the colonic response to DSS challenge in WT and NHE3^−/− mice. In NHE3^−/− mice, broad-spectrum oral antibiotics or anti-asialo GM1 antibodies reduce the expression of IFN-γ and iNOS to basal levels and delay but do not prevent severe mortality in response to DSS treatment. Conclusions These results suggest that NHE3 participates in mucosal responses to epithelial damage, acting as a modifier gene determining the extent of the gut inflammatory responses in the face of intestinal injury.

We sought to characterize expression of an apically expressed intestinal Na-P_icotransporter (Na-P_i-IIb) during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na-P_iuptake by intestinal brush-border membrane vesicles was highest at 14 days of age, lower at 21 days, and further reduced at 8 wk and 8–9 mo of age. Na-P_i-IIb mRNA and immunoreactive protein levels in 14-day-old animals were markedly higher than in older groups. MP treatment significantly decreased Na-P_iuptake and Na-P_i-IIb mRNA and protein expression in 14-day-old mice. Additionally, the size of the protein was smaller in 14-day-old mice. Deglycosylation of protein from 14-day-old and 8-wk-old animals with peptide N-glycosidase reduced the molecular weight to the predicted size. We conclude that intestinal Na-P_iuptake and Na-P_i-IIb expression are highest at 14 days and decrease with age. Furthermore, MP treatment reduced intestinal Na-P_iuptake approximately threefold in 14-day-old mice and this reduction correlates with reduced Na-P_i-IIb mRNA and protein expression. We also demonstrate that Na-P_i-IIb is an N-linked glycoprotein and that glycosylation is age dependent.