Suppressors of cytokine signaling (SOCS) provide negative regulation of inflammatory reaction. The role and precise cellular mechanisms of SOCS1 in control of endothelial dysfunction and barrier compromise associated with acute lung injury remain unexplored. Our results show that siRNA-mediated SOCS1 knockdown augmented lipopolysaccharide (LPS)-induced pulmonary endothelial cell (EC) permeability and enhanced inflammatory response. Consistent with in vitro data, EC-specific SOCS1 knockout mice developed more severe lung vascular leak and accumulation of inflammatory cells in bronchoalveolar lavage fluid. SOCS1 overexpression exhibited protective effects against LPS-induced endothelial permeability and inflammation, which were dependent on microtubule (MT) integrity. Biochemical and image analysis of unstimulated EC showed SOCS1 association with the MT, while challenge with LPS or MT depolymerizing agent colchicine impaired this association. SOCS1 directly interacted with N2 domains of MT-associated proteins CLIP-170 and CLASP2. Furthermore, N-terminal region of SOCS1 was indispensable for these interactions and SOCS1-ΔN mutant lacking N-terminal 59 amino acids failed to rescue LPS-induced endothelial dysfunction. Depletion of endogenous CLIP-170 or CLASP2 abolished SOCS1 interaction with Toll-like receptor-4 and Janus kinase-2 leading to impairment of SOCS1 inhibitory effects on LPS-induced inflammation. Altogether, these findings suggest that endothelial barrier protective and anti-inflammatory effects of SOCS1 are critically dependent on its targeting to the MT.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood death from diarrhea and the leading cause of Traveler’s diarrhea. E. coli heat-stable enterotoxin (ST) is a major virulence factor of ETEC and inhibits the brush border Na/H exchanger NHE3 in producing diarrhea. NHE3 regulation involves multiprotein signaling complexes that form on its COOH terminus. In this study, the hypothesis was tested that ST signals via members of the Na/H exchanger regulatory factor (NHERF) family of scaffolding proteins, NHERF2, which had been previously shown to have a role, and now with concentration on a role for NHERF3. Two models were used: mouse small intestine and Caco-2/BBe cells. In both models, ST rapidly increased intracellular cGMP, inhibited NHE3 activity, and caused a quantitatively similar decrease in apical expression of NHE3. The transport effects were NHERF3 and NHERF2 dependent. Also, mutation of the COOH-terminal amino acids of NHERF3 supported that NHERF3-NHERF2 heterodimerization was likely to account for this dual dependence. The ST increase in cGMP in both models was partially dependent on NHERF3. The intracellular signaling pathways by which ST-cGMP inhibits NHE3 were different in mouse jejunum (activation of cGMP kinase II, cGKII) and Caco-2 cells, which do not express cGKII (elevation of intracellular Ca2+ concentration [Ca2+]i). The ST elevation of [Ca2+]i was from intracellular stores and was dependent on NHERF3-NHERF2. This study shows that intracellular signaling in the same diarrheal model in multiple cell types may be different; this has implications for therapeutic strategies, which often assume that models have similar signaling mechanisms.

Autosomal-dominant polycystic kidney disease (ADPKD) induces a secretory phenotype, resulting in multiple fluid-filled cysts. We have previously demonstrated that VX-809, a corrector of the cystic fibrosis transmembrane conductance regulator (CFTR), reduces cyst growth. Here, we show that in normal mice CFTR is located within the cells and also at the apical and basolateral membranes. However, in polycystic kidney disease (pkd1)-knockout mice, CFTR was located at the plasma membrane, consistent with its role in cAMP-dependent fluid secretion. In cystic mice, VX-809 treatment increased CFTR levels at the apical membrane and reduced its association with the endoplasmic reticulum. Surprisingly, VX-809 treatment significantly increased CFTR's co-localization with the basolateral membrane in cystic mice. Na+/H+ exchanger 3 (NHE3) is present in pkd1-knockout and normal mice and in proximal tubule-derived, cultured pkd1-knockout cells. VX-809 increased the expression, activity, and apical plasma membrane localization of NHE3. Co-localization of epithelial sodium channel (ENaC) with the plasma membrane was reduced in cysts in pkd1-knockout mice, consistent with an inability of the cysts to absorb fluid. Interestingly, in the cystic mice, VX-809 treatment increased ENaC levels at the apical plasma membrane consistent with fluid absorption. Thus, VX-809 treatment of pkd1-null mouse kidneys significantly affected CFTR, NHE3, and ENaC, altering the cyst phenotype from one poised toward fluid secretion toward one more favorable for absorption. VX-809 also altered the location of CFTR but not of NHE3 or ENaC in normal mice. Given that VX-809 administration is safe, it may have potential utility for treating patients with ADPKD.

maintained remission in the long-term follow-up by identifying the trigger foods.Topical steroids seem to have a higher efficacy in the short-term, but requires repeated cycles to maintain long-term remission.

The investigation of orphan GPCRs (GPRs) has the potential to uncover novel insights into whole animal physiology. In this study, our goal was to determine the renal localization of Gprc5c, a receptor that we previously reported to be highly expressed in murine whole kidney, and to examine physiologic parameters in Gprc5c knockout (KO) mice to gain insight into function. Gprc5c localized to the apical membrane of renal proximal tubules (PTs) in mice, rats, and humans. With the comparison of Gprc5c wild-type (WT) and KO mice, we found that Gprc5c KO mice have altered acid-base homeostasis. Specifically, Gprc5c KO mice have lower blood pH and higher urine pH compared with WT mice, with a reduced level of titratable acids in their urine. In an in vitro GPCR internalization assay, we observed that Gprc5c internalization (an index of activation) was triggered by alkaline extracellular pH. Furthermore, with the use of an in vitro BCECF assay, we observed that Gprc5c increases Na+/H+ exchanger 3 (NHE3) activity at alkaline pH. We also find that the NHE3 activity is reduced in Gprc5c KO mice by 2 photon imaging in seminaphthorhodafluors (SNARF)-4F-loaded kidney sections. NHE3 is a primary contributor to apical transport of H+ in the renal PT. Together, these data imply that Gprc5c modulates the renal contribution to systemic pH homeostasis, at least in part, by taking part in the regulation of NHE3.

Casein kinase 2 (CK2) binds to the NHE3 C-terminus and constitutively phosphorylates a downstream site (S719) that accounts for 40% of basal NHE3 activity. The role of CK2 in regulation of NHE3 activity in polarized Caco-2/bbe cells was further examined by mutation of NHE3-S-719 to A (not phosphorylated) or D (phosphomimetic). NHE3-S719A but not -S719D had multiple changes in NHE3 activity: 1) reduced basal NHE3 activity-specifically, inhibition of the PI3K/AKT-dependent component; 2) reduced acute stimulation of NHE3 activity by LPA/LPA5R stimulation; and 3) reduced acute inhibition of NHE3 activity-specifically, elevated Ca2+ related (carbachol/Ca2+ ionophore), but there was normal inhibition by forskolin and hyperosmolarity. The S719A mutant had reduced NHE3 complex size, reduced expression in lipid rafts, increased BB mobile fraction, and reduced binding to multiple proteins that bind throughout the NHE3 intracellular C-terminus, including calcineurin homologous protein, the NHERF family and SNX27 (related PDZ domains). These studies show that phosphorylation of the NHE3 at a single amino acid in the distal part of the C-terminus affects multiple aspects of NHE3 complex formation and changes the NHE3 lipid raft distribution, which cause changes in specific aspects of basal as well as acutely stimulated and inhibited Na+/H+ exchange activity.

Recent studies have identified orphan G‐protein coupled receptors (GPRs) as sensors of physiological metabolites. In this study, our goal is to elucidate the functional role of an understudied renal GPR, Gprc5c, which we previously found to be highly expressed in the kidney (Rajkumar et al., 2014). By immunofluorescence (IF), we find that Gprc5c localizes to the apical renal proximal tubules (PTs) in wild‐type (WT) mice, where it colocalizes with two PT markers ‐ LTA & megalin. Staining is absent in Gprc5c knockout (KO) mice kidneys. Gprc5c also localizes to the apical PTs by immunofluorescence in both rats and humans, suggesting an evolutionarily conserved role. In vivo , Gprc5c KO were not different from WT with respect to blood pressure, kidney/body weight, blood levels of Na + , Cl − & glucose, and urine levels of creatinine & glucose. However, Gprc5c KO have higher urine pH (WT: 5.48±0.01, n=6; KO: 5.81±0.06, n=6; p<0.05). In addition, the blood pH of Gprc5c KO mice trends acidic compared to WT in unanaesthetized mice (WT: 7.44±0.04, n=4; KO: 7.34±0.02, n=4; p=0.07), and in mice under isofluorane (WT: 7.22±0.03, n=5; KO: 7.03±0.04, n=5; p<0.05). In an in vitro GPCR internalization assay, alkaline pH (but not other chemicals tested) triggered Gprc5c internalization (an index of activation), with maximal internalization seen above pH 7.4 (pH 8.0>pH 7.4>pH 6.5). Other GPCRs tested as controls showed no changes in internalization over the pH range tested. On the apical membrane of the renal PT, sodium hydrogen exchanger 3 (NHE3) plays a key role in bicarbonate reabsorption, and therefore in maintaining pH homeostasis. Thus, we examined whether Gprc5c alters sodium proton exchanger activity. Using BCECF in a ratiometric fluorescence assay in Hek293T cells, we find that Gprc5c increases sodium proton exchange by 1.6 fold compared to empty vector controls in alkaline conditions (pH 8.0) (p<0.05), but not at pH 7.4. The increase seen at pH 8 is Tenapanor‐sensitive, implying that it is mediated via NHE3. In preliminary data, we also find that Gprc5c KO mice exhibit blunted changes in urinary pH when challenged with an alkali load for 7 days. Together, these data imply that Gprc5c localizes to the renal PTs apically where it contributes to systemic pH homeostasis via modulation of NHE3 function.

NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to the NHE3 C-terminal four amino acids. The internal NHERF-binding region contains both putative Class I (-(592)SAV-) and Class II (-(CLDM)-C-595-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminal PBM mutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determines NHE3 stability in the PM, and the latter determines total expression and percent PM expression.

this P-gp promoter construct (p-428/+703) harbors the HNF1β response element(s).Interestingly, sequence analysis of this region revealed the presence of one potential HNF1 binding site.Additionally, ChIP analysis utilizing primers spanning the p-428/+703 region showed markedly enhanced association of HNF1β (~9 fold) with endogenous Pgp promoter in Caco-2 cells.In conclusion, these studies demonstrate that HNF1β directly upregulates P-gp gene expression.We speculate that this increase in intestinal P-gp expression may underlie the protective effect of HNF1β in maintaining intestinal epithelial integrity.

Na+/H+ exchanger NHE3 mediates the majority of intestinal and renal electroneutral sodium absorption. Dysfunction of NHE3 is associated with a variety of diarrheal diseases. We previously reported that the NHE3 gene (SLC9A3) has more than 400 single-nucleotide polymorphisms (SNPs) but few nonsynonymous polymorphisms. Among the latter, one polymorphism (rs2247114-G>A), which causes a substitution from arginine to cysteine at amino acid position 799 (p.R799C), is common in Asian populations. To improve our understanding of the population distribution and potential clinical significance of the NHE3-799C variant, we investigated the frequency of this polymorphism in different ethnic groups using bioinformatics analyses and in a cohort of Japanese patients with cardiovascular or renal disease. We also characterized the function of human NHE3-799C and its sensitivity to regulatory ligands in an in vitro model. NHE3-799C had an allele frequency of 29.5-57.6% in Asian populations, 11.1-23.6% in European populations, and 10.2-22.7% in African populations. PS120/FLAG-NHERF2 fibroblasts stably expressing NHE3-799C had lower total protein expression but a higher percentage of surface expression than those expressing NHE3-799R. NHE3-799C had similar basal activity to NHE3-799R and was similarly stimulated or inhibited, by serum or forskolin, respectively. Tenapanor, a small-molecule NHE3 inhibitor, dose-dependently inhibited NHE3-799R and NHE3-799C activities. The IC50 values of tenapanor for NHE3-799C and NHE3-799R were significantly different, but both were in the nanomolar range. These results suggest that NHE3-799C is a common variant enriched in Asian populations, is not associated with compromised function or abnormal regulation, and is unlikely to contribute to clinical disease.NEW & NOTEWORTHY This study reports results on the functional significance of human NHE3-799C under basal conditions and in response to regulatory ligands, including a novel NHE3 inhibitor called tenapanor. We demonstrate that NHE3-799C is a common variant of NHE3 that is enriched in Asian populations; however, in contrast to our previous studies using rabbit NHE3, its presence seems to have limited clinical significance in humans and is not associated with compromised function or abnormal transport regulation.

Recent studies have found that novel G‐protein coupled receptors (GPCRs) are utilized as specialized chemosensors to detect metabolites in a variety of tissues, and are often involved in mediating essential physiological processes and metabolic pathways. Our lab aims to uncover the functional role of novel GPCRs in the kidney, and to that end we recently used a Taqman‐based mouse GPCR array to identify GPCRs with unknown function that are expressed at high levels in murine kidney (Rajkumar, et al PLoS One 2014). In this study, we focused on elucidating the functional role of one such novel GPCR, Gprc5c. Gprc5c is an orphan GPCR with no known ligand which we found to be present in the kidney by real‐time PCR at a comparable level to the well‐studied and highly expressed Angiotensin receptor, AT1a (ΔCt AT1a : 6.25±0.24; ΔCt Gprc5c : 7.0±0.16). Using immunohistochemistry, we found that Gprc5c localizes to the renal proximal tubule apical brush border in wild‐type (WT) mice, where it colocalized with both LTA (lotus tetragonolobus agglutinin) and megalin (Gprc5c staining was absent in whole‐animal Gprc5c knockout (KO) mice). Because Gprc5c is an orphan receptor with no known ligand, we performed ligand‐induced GPCR internalization assays in vitro to identify activators of Gprc5c in transiently transfected HEK293T cells. We found that alkaline pH triggered Gprc5c activation, with maximal internalization seen above pH 7.4 (pH 8.0>pH 7.4>pH 6.5). Importantly, other GPCRs tested as controls showed no changes in internalization over the pH range tested. In agreement with a role for Gprc5c in responding to changes in extracellular pH, we found that Gpr5c KO mice have alkaline urine pH compared to wild‐type littermates (WT: 5.5±0.02, n=4; KO: 5.76±0.05, n=5; p<0.05). In addition, preliminary studies indicate that the plasma pH of Gprc5c knockout mice trends mildly acidic (WT: 7.44±0.04, n=4; KO: 7.34±0.02, n=4; p=0.07). To begin to understand the mechanism by which Gprc5c may modulate pH regulation, we loaded HEK293T cells with BCECF‐AM and determined that Gprc5c increases the activity and set point of NHE3 (a sodium‐hydrogen exchanger expressed in the brush border of the renal proximal tubule). Under alkaline conditions (pH8), we find that Gprc5c increases sodium proton exchange by 1.6 fold compared to control cells (p<0.05), and that this increase in activity is Tenapanor‐sensitive, implying that it is mediated via NHE3. Together, these results demonstrate that Gprc5c localizes to the renal proximal tubule where it contributes to pH homeostasis via modulation of NHE3 function.

SERT mRNA was further investigated.Decay of SERT mRNA was measured in the presence of transcription inhibitor actinomycin D. Interestingly, cGMP decreased the half-life of SERT variant from 23.5 ± 2.36 h to 14.51 ± 1.5 h (P<0.01).The decrease in SERT mRNA expression was replicated in the mice Ileal organoids treated with cGMP (~65% inhibition at 24h). Conclusion:Our results demonstrate that cGMP inhibits intestine specific SERT variant by decreasing the transcript stability.These data highlight novel mechanisms by which cGMP alters serotonergic machinery in the IEC that may be of relevance in GI disorders such as infectious diarrhea or IBS.

Genetic determinants appear to play a role in susceptibility to chronic diarrhea, but the genetic abnormalities involved have only been identified in a few conditions. The Na+/H+ exchanger 3 (NHE3) accounts for a large fraction of physiologic intestinal Na+ absorption. It is highly regulated through effects on its intracellular COOH-terminal regulatory domain. The impact of genetic variation in the NHE3 gene, such as single nucleotide polymorphisms (SNPs), on transporter activity remains unexplored. From a total of 458 SNPs identified in the entire NHE3 gene, we identified three nonsynonymous mutations (R474Q, V567M, and R799C), which were all in the protein's intracellular COOH-terminal domain. Here we evaluated whether these SNPs affect NHE3 activity by expressing them in a mammalian cell line that is null for all plasma membrane NHEs. These variants significantly reduced basal NHE3 transporter activity through a reduction in intrinsic NHE3 function in variant R474Q, abnormal trafficking in variant V567M, or defects in both intrinsic NHE3 function and trafficking in variant R799C. In addition, variants NHE3 R474Q and R799C failed to respond to acute dexamethasone stimulation, suggesting cells with these mutant proteins might be defective in NHE3 function during postprandial stimulation and perhaps under stressful conditions. Finally, variant R474Q was shown to exhibit an aberrant interaction with calcineurin B homologous protein (CHP), an NHE3 regulatory protein required for basal NHE3 activity. Taken together, these results demonstrate decreased transport activity in three SNPs of NHE3 and provide mechanistic insight into how these SNPs impact NHE3 function.

Sorting nexin 27 (SNX27) contains a PDZ domain that is phylogenetically related to the PDZ domains of the NHERF proteins. Studies on nonepithelial cells have shown that this protein is located in endosomes, where it regulates trafficking of cargo proteins in a PDZ domain-dependent manner. However, the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) primarily through the SNX27 PDZ domain. A combination of knockdown and reconstitution experiments with wild type and a PDZ domain mutant (GYGF → GAGA) of SNX27 demonstrate that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation-based recycling and degradation studies in intestinal epithelial cells show that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ-mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells.

The Na(+)/H(+) exchanger 3 (NHE3) is a brush border (BB) Na(+)/H(+) antiporter that accounts for the majority of physiologic small intestinal and renal Na(+) absorption. It is regulated physiologically and in disease via changes in endocytosis/exocytosis. Paradoxically, NHE3 is fixed to the microvillar (MV) actin cytoskeleton and has little basal mobility. This fixation requires NHE3 binding to the multi-PDZ domain scaffold proteins Na(+)/H(+) exchanger regulatory factor (NHERF)1 and NHERF2 and to ezrin. Coordinated release of NHE3 from the MV cytoskeleton has been demonstrated during both stimulation and inhibition of NHE3. However, the signaling molecules involved in coordinating NHE3 trafficking and cytoskeletal association have not been identified. This question was addressed by studying lysophosphatidic acid (LPA) stimulation of NHE3 in polarized renal proximal tubule opossum kidney (OK) cells that occurs via apical LPA5 receptors and is NHERF2 dependent and mediated by epidermal growth factor receptor (EGFR), Rho/Rho-associated kinase (ROCK), and ERK. NHE3 activity was determined by BCECF/fluorometry and NHE3 microvillar mobility by FRAP/confocal microscopy using NHE3-EGFP. Apical LPA (3 μM)/LPA5R stimulated NHE3 activity, increased NHE3 mobility, and decreased the NHE3/NHERF2 association. The LPA stimulation of NHE3 was also PKCδ dependent. PKCδ was necessary for LPA stimulation of NHE3 mobility and NHE3/NHERF2 association. Moreover, the LPA-induced translocation to the membrane of PKCδ was both ERK and phospholipase C dependent with ERK acting upstream of PLC. We conclude that LPA stimulation of NHE3 exocytosis includes a signaling pathway that regulates fixation of NHE3 to the MV cytoskeleton. This involves a signaling module consisting of ERK-PLC-PKCδ, which dynamically and reversibly releases NHE3 from NHERF2 to contribute to the changes in NHE3 MV mobility.