Sel1-like proteins and peptides are the major Oxalobacter formigenes-derived factors stimulating oxalate transport by human intestinal epithelial cells.

Kidney stones (KSs) are very common, excruciating, and associated with tremendous healthcare cost, chronic kidney disease (CKD), and kidney failure (KF). Most KSs are composed of calcium oxalate and small increases in urinary oxalate concentration significantly enhance the stone risk. Oxalate also potentially contributes to CKD progression, kidney disease-associated cardiovascular diseases, and poor renal allograft survival. This emphasizes the urgent need for plasma and urinary oxalate lowering therapies, which can be achieved by enhancing enteric oxalate secretion. We previously identified Oxalobacter formigenes (O. formigenes)-derived factors secreted in its culture-conditioned medium (CM), which stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells and reduce urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion. Given their remarkable therapeutic potential, we now identified Sel1-like proteins as the major O. formigenes-derived secreted factors using mass spectrometry and functional assays. Crystal structures for six proteins were determined to confirm structures and better understand functions. OxBSel1-14-derived small peptides P8 and P9 were identified as the major factors, with P8 + 9 closely recapitulating the CM's effects, acting through the oxalate transporters SLC26A2 and SLC26A6 and PKA activation. Besides C2 cells, P8 + 9 also stimulate oxalate transport by human ileal and colonic organoids, confirming that they work in human tissues. In conclusion, P8 and P9 peptides are identified as the major O. formigenes-derived secreted factors and they have significant therapeutic potential for hyperoxalemia, hyperoxaluria, and related disorders, impacting the outcomes of patients suffering from KSs, enteric hyperoxaluria, primary hyperoxaluria, CKD, KF, and renal transplant recipients.NEW & NOTEWORTHY We previously identified Oxalobacter formigenes-derived secreted factors stimulating oxalate transport by human intestinal epithelial cells in vitro and reducing urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion. We now identified Sel1-like proteins and small peptides as the major secreted factors and they have significant therapeutic potential for hyperoxalemia and hyperoxaluria, impacting the outcomes of patients suffering from kidney stones, primary and secondary hyperoxaluria, chronic kidney disease, kidney failure, and renal transplant recipients.

Gnotobiotic Operations and Assembly for Development of Germ-Free Animal Model of Laser-Induced Choroidal Neovascularization.

Purpose: Compelling new evidence reveals a close link between the gut microbiome and the pathogenesis of neovascular age-related macular degeneration (nAMD). Germ-free (GF) animal models are the current gold standard for studying host the microbe interactions in vivo; yet, no GF animal models of nAMD are available today. This protocol describes gnotobiotic operations and assembly for a laser-induced choroidal neovascularization (CNV) model in GF mice to study the gut microbiome in neovascular AMD. Methods: We developed a step-wise approach to performing retinal laser photocoagulation in GF C57BL/6J mice that were bred and maintained at the gnotobiotic facility. Following a strict sterility protocol, we administered laser photocoagulation via an Argon 532-nm laser attached to a customized slit-lamp delivery system. Sterility was confirmed by weekly fecal cultures and reverse transcriptase-polymerase chain reaction. Results: The experiment was repeated twice at different time points using seven mice (14 eyes). Stool cultures and RT-PCR remained negative for 14 days post-procedure in all mice. Lectin immunostaining performed on choroidal flatmounts confirmed the presence of CNV lesions 2 weeks after laser treatment. Conclusions: We established a GF mouse model of nAMD with detailed guidelines to deliver retinal laser in GF mice maintaining sterility after the laser procedure. Translational Relevance: To our knowledge, this is the first protocol that describes a GF murine model of laser-induced CNV. In addition to nAMD, this animal model can be used to investigate host-microbial interactions in other eye diseases with laser-induced mouse models such as glaucoma and retinal vein occlusion.

Activation of the PKA signaling pathway stimulates oxalate transport by human intestinal Caco2-BBE cells.

Most kidney stones are composed of calcium oxalate, and small increases in urine oxalate enhance the stone risk. The mammalian intestine plays a crucial role in oxalate homeostasis, and we had recently reported that Oxalobacter-derived factors stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells through PKA activation. We therefore evaluated whether intestinal oxalate transport is directly regulated by activation of the PKA signaling pathway. To this end, PKA was activated with forskolin and IBMX (F/I). F/I significantly stimulated (3.7-fold) [14C]oxalate transport by C2 cells [≥49% of which is mediated by the oxalate transporter SLC26A6 (A6)], an effect completely blocked by the PKA inhibitor H89, indicating that it is PKA dependent. PKA stimulation of intestinal oxalate transport is not cell line specific, since F/I similarly stimulated oxalate transport by the human intestinal T84 cells. F/I significantly increased (2.5-fold) A6 surface protein expression by use of immunocytochemistry. Assessing [14C]oxalate transport as a function of increasing [14C]oxalate concentration in the flux medium showed that the observed stimulation is due to a F/I-induced increase (1.8-fold) in Vmax and reduction (2-fold) in Km. siRNA knockdown studies showed that significant components of the observed stimulation are mediated by A6 and SLC26A2 (A2). Besides enhancing A6 surface protein expression, it is also possible that the observed stimulation is due to PKA-induced enhanced A6 and/or A2 transport activity in view of the reduced Km. We conclude that PKA activation positively regulates oxalate transport by intestinal epithelial cells and that PKA agonists might therapeutically impact hyperoxalemia, hyperoxaluria, and related kidney stones.

Oxalobacter formigenes-Derived Bioactive Factors Stimulate Oxalate Transport by Intestinal Epithelial Cells.

Hyperoxaluria is a major risk factor for kidney stones and has no specific therapy, although Oxalobacter formigenes colonization is associated with reduced stone risk. O. formigenes interacts with colonic epithelium and induces colonic oxalate secretion, thereby reducing urinary oxalate excretion, via an unknown secretagogue. The difficulties in sustaining O. formigenes colonization underscore the need to identify the derived factors inducing colonic oxalate secretion. We therefore evaluated the effects of O. formigenes culture conditioned medium (CM) on apical 14C-oxalate uptake by human intestinal Caco-2-BBE cells. Compared with control medium, O. formigenes CM significantly stimulated oxalate uptake (>2.4-fold), whereas CM from Lactobacillus acidophilus did not. Treating the O. formigenes CM with heat or pepsin completely abolished this bioactivity, and selective ultrafiltration of the CM revealed that the O. formigenes-derived factors have molecular masses of 10-30 kDa. Treatment with the protein kinase A inhibitor H89 or the anion exchange inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid completely blocked the CM-induced oxalate transport. Knockdown of the oxalate transporter SLC26A6 also significantly restricted the induction of oxalate transport by CM. In a mouse model of primary hyperoxaluria type 1, rectal administration of O. formigenes CM significantly reduced (>32.5%) urinary oxalate excretion and stimulated (>42%) distal colonic oxalate secretion. We conclude that O. formigenes-derived bioactive factors stimulate oxalate transport in intestinal cells through mechanisms including PKA activation. The reduction in urinary oxalate excretion in hyperoxaluric mice treated with O. formigenes CM reflects the in vivo retention of biologic activity and the therapeutic potential of these factors.

Cyclic AMP-mediated endocytosis of intestinal epithelial NHE3 requires binding to synaptotagmin 1.

The apical membrane Na(+)-H(+) exchanger (NHE)3 is regulated by cAMP-dependent phosphorylation, which inhibits its activity through membrane endocytosis. The clathrin complex adaptor protein synaptotagmin 1 (Syt 1) appears to be essential to this process, but little is known about its expression in intestinal epithelial cells or interaction with NHE3. The intestinal epithelial expression and apical location of Syt 1 were determined by Syt 1 mRNA profiling and immunolocalization. Tandem mass spectrometry was used for protein identification. Bis(sulfosuccinimidyl) suberate (BS(3)) cross linking suggested that NHE3 and Syt 1 were in a membrane complex following cAMP stimulation of Caco2BBE (Brush Border Expressions) cells. To investigate the regulation of NHE3 appearance in a Syt 1-containing membrane compartment, doxycycline-inducible hemaglutinin (HA)-tagged NHE3 was expressed in Caco2BBE cells. HA-NHE3 correctly targeted to the apical membrane, where, upon cAMP stimulation, it was internalized with a Syt 1-containing compartment. Site-directed mutagenesis of NHE3 showed that serine 605 (S605) was pivotal to NHE3 and Syt 1 association and internalization. Direct Syt 1 interaction with NHE3 was suggested by fluorescence resonance energy transfer (FRET) analysis. The physiological role of S552 was less clear. By FRET, this serine residue appeared to be involved in cAMP-induced Syt 1 binding of NHE3. However, when HA-tagged NHE3 S552A was expressed in Caco2 cells, the mutated construct was not inserted into the apical membrane. We conclude that intestinal epithelial Syt 1 plays an important role in cAMP-stimulated endocytosis of apical NHE3 through cAMP-dependent phosphorylation of S605 that is required for NHE3 and Syt 1 association.

Alpha2-adrenergic receptors attenuate secretagogue-induced endocytosis and promote exocytosis of intestinal NHE2 and NHE3.

Adrenergic agonists, through activation of intestinal epithelial alpha2-adrenergic receptors (alpha2AR), inhibit electrolyte secretion and promote absorption. The mechanisms of action to promote basal Na(+) absorption and inhibit stimulated secretion are not understood completely. The effects of alpha2-agonists on Na(+) transport were studied in a cell line, Caco2-3B, derived from the Caco2 cell line engineered to permanently express human alpha2A-adrenergic receptors. Serosal, but not mucosal, addition of the alpha2AR agonist N-(2,6-dichlorophenyl)-4,5-dihydro-1H-imidazol-2-amine (clonidine) increased Caco2-3B apical (22)Na(+) uptake, an effect not seen in the Caco2 parent line that lacks alpha2AR expression. This effect was blocked by the alpha2AR antagonist 17alpha-yohmban-16alpha-carboxylic acid methyl ester (yohimbine). Increased Na(+) uptake was paralleled by increased apical surface abundance of the sodium/hydrogen exchangers NHE2 and NHE3. No changes in total cell NHE2 and NHE3 expression were observed. Clonidine also inhibited both cAMP and Ca(2+)-induced decreases in apical Na(+) uptake and apical membrane NHE2 and NHE3 endocytosis stimulated by these agents. alpha2AR actions were mediated via stimulation of phospholipase C, and metabolism of arachidonic acid by an epoxygenase activity followed epidermal growth factor release and activation of the epidermal growth factor receptor, resulting in phosphatidylinositol-3-kinase and Akt stimulation. In summary, activation of intestinal epithelial alpha2AR significantly blocks the inhibition of apical Na(+) transporters by cAMP- and Ca(2+)-mediated pathways and also directly increases apical sodium/hydrogen exchange activities. By both blocking electrolyte secretion and promoting absorption, alpha2-agonists could be potent antidiarrheal agents that could directly counteract the actions of toxigenic pathogens and other secretagogues causing secretory diarrhea.

Functional coupling of the downregulated in adenoma Cl-/base exchanger DRA and the apical Na+/H+ exchangers NHE2 and NHE3.

Non-nutrient-dependent salt absorption across the brush-border membrane of intestinal epithelial cells is primarily mediated by coupled apical Na(+)/H(+) (aNHE) and anion exchange transport, with the latter suspected of being mediated by DRA (downregulated in adenoma; SLC26A3) that is defective in congenital chloridorrhea. To investigate DRA in greater detail and determine whether DRA and NHE activities can be coupled, we measured (22)Na(+) and (36)Cl(-) uptake in Caco2BBE colon cells infected with the tet-off-inducible DRA transgene. Under basal conditions, DRA activity was low in normal and infected Caco2BBE cells in the presence of tetracycline, whereas NHE activities could be easily detected. When apical NHE activity was increased by transfection or serum-induced expression of the aNHE isoforms NHE2 and NHE3, increased (36)Cl(-) uptake was observed. Inhibition of DRA activity by niflumic acid was greater than that by DIDS as well as by the NHE inhibitor dimethylamiloride and the carbonic anhydrase inhibitor methazolamide. DRA activity was largely aNHE-dependent, whereas a component of DRA-independent aNHE uptake continued to be observed. Coupled aNHE and DRA activities were inhibited by increased cellular cAMP and calcium and were associated with synaptotagmin I-dependent, clathrin-mediated endocytosis. In summary, these data support the role of DRA in electroneutral NaCl absorption involving functional coupling of Cl(-)/base exchange and apical NHE.

Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP- and Ca2+-induced endocytosis by recruitment of AP2 and clathrin.

Apical membrane sodium hydrogen exchanger 3 (NHE3), a major pathway for non-nutrient-dependent intestinal Na(+) absorption, is tightly regulated by second messenger systems that affect its functional activity and membrane trafficking. However, the events and components involved in NHE3 regulation are only partially understood. We report that the adaptor protein synaptotagmin I (Syt I) plays a pivotal role in cAMP- and Ca(2+)-induced cargo recognition of NHE3 and initiation of its endocytosis. Both mouse small intestine (jejunum) and Caco-2BBe Syt I coimmunoprecipitated with NHE3, particularly following increases in cellular cAMP or Ca(2+). Following short interfering RNA (siRNA) suppression of Syt I expression, cAMP- and Ca(2+)-induced inhibition of NHE3 activity were still observed but NHE3 endocytosis was blocked, as assessed by (22)Na influx and apical membrane biotin labeling, respectively. Similar effects on NHE3 inhibition and endocytosis were observed by siRNA suppression of either the mu-subunit of the adaptor protein 2 (AP2) complex or the heavy chain of clathrin. Coimmunoprecipitation analyses of NHE3 with these adaptor proteins revealed that cAMP- and Ca(2+)-induced NHE3-Syt I interaction preceded and was required for recruitment of AP2 and the clathrin complex. Confocal microscopy confirmed both the time sequence and protein associations of these events. We conclude that Syt I plays a pivotal role in mediating cAMP- and Ca(2+)-induced endocytosis of NHE3 (but not in inhibition of activity) through cargo recognition of NHE3 and subsequent recruitment of AP2-clathrin assembly required for membrane endocytosis.

fMLP induces Hsp27 expression, attenuates NF-kappaB activation, and confers intestinal epithelial cell protection.

Sustained expression of cytoprotective intestinal epithelial heat shock proteins (Hsps), particularly Hsp27, depends on stimuli derived from bacterial flora. In this study, we examined the role of the bacterial chemotactic peptide fMLP in stimulating colonic epithelial Hsp expression at concentrations encountered in a physiological milieu. Treatment of the polarized human intestinal epithelial cell line Caco2bbe with physiological concentrations of fMLP (10-100 nM) induced expression of Hsp27, but not Hsp72, in a time- and concentration-dependent manner. Induction of Hsp27 by fMLP was specific since the fMLP analogs MRP and MLP were not effective. Hsp27 induction by fMLP was blocked by the fMLP-receptor antagonist BOC-FLFLF and was blocked when the dipeptide transporter PepT1, an entry pathway for fMLP, was silenced. fMLP activated both the p38 and ERK1/2 MAP kinase pathways in Caco2bbe cells, but not the SAPK/JNK pathway. The p38 inhibitor SB203580, but not the MEK-1 inhibitor PD98059, blocked Hsp27 induction by fMLP. fMLP treatment inhibited actin depolymerization and decreased transepithelial resistance caused by the oxidant monochloramine, and this inhibition was reversed by silencing Hsp27 expression. fMLP pretreatment also inhibited activation of proinflammatory transcription factor NF-kappaB by TNF-alpha in Caco2bbe cells, reducing induction of NF-kappaB target genes by TNF-alpha both in human intestinal biopsies and Caco2bbe cells. In conclusion, fMLP may contribute to the maintenance of intestinal homeostasis by mediating physiological expression of Hsp27, enhancing cellular protection, and negatively regulating the inflammatory response.

Luminal bacterial flora determines physiological expression of intestinal epithelial cytoprotective heat shock proteins 25 and 72.

Heat shock proteins (HSP) 25 and 72 are expressed normally by surface colonocytes but not by small intestinal enterocytes. We hypothesized that luminal commensal microflora maintain the observed colonocyte HSP expression. The ability of the small intestine to respond to bacteria and their products and modulate HSPs has not been determined. The effects of luminal bacterial flora in surgically created midjejunal self-filling (SFL) vs. self-emptying (SEL) small-bowel blind loops on epithelial HSP expression were studied. HSP25 and HSP72 expression were assessed by immunoblot and immunohistochemistry. SFL were chronically colonized, whereas SEL contained levels of bacteria normal for the proximal small intestine. SFL creation significantly increased HSP25 and HSP72 expression relative to corresponding sections from SEL. Metronidazole treatment, which primarily affects anaerobic bacteria as well as a diet lacking fermentable fiber, significantly decreased SFL HSP expression. Small bowel incubation with butyrate ex vivo induced a sustained and significant upregulation of HSP25 and altered HSP72 expression, confirming the role of short-chain fatty acids. To determine whether HSPs induction altered responses to an injury, effects of the oxidant, monochloramine, on epithelial resistance and short-circuit current (I(sc)) responses to carbachol and glucose were compared. Increased SFL HSP expression was associated with protection against oxidant-induced decreases in transmural resistance and I(sc) responses to glucose, but not secretory responses to carbachol. In conclusion, luminal microflora and their metabolic byproducts direct expression of HSPs in gut epithelial cells, an effect that contributes to preservation of epithelial cell viability under conditions of stress.