TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.

Oxidative damage to renal tubular epithelial cells is a fundamental pathogenic mechanism implicated in both acute kidney injury and chronic kidney diseases. Because epithelial cell survival influences the outcome of acute kidney injury and chronic kidney diseases, identifying its molecular regulators could provide new insight into pathobiology and possible new therapeutic strategies for these diseases. We have identified transmembrane and immunoglobulin domain-containing 1 (TMIGD1) as a novel adhesion molecule, which is highly conserved in humans and other species.

Glucose-dependent insulinotropic polypeptide-mediated signaling pathways enhance apical PepT1 expression in intestinal epithelial cells.

We have shown recently that glucose-dependent insulinotropic polypeptide (GIP), but not glucagon-like peptide 1 (GLP-1) augments H(+) peptide cotransporter (PepT1)-mediated peptide absorption in murine jejunum. While we observed that inhibiting cAMP production decreased this augmentation of PepT1 activity by GIP, it was unclear whether PKA and/or other regulators of cAMP signaling pathway(s) were involved. This study utilized tritiated glycyl-sarcosine [(3)H-glycyl-sarcosine (Gly-Sar), a relatively nonhydrolyzable dipeptide] uptake to measure PepT1 activity in CDX2-transfected IEC-6 (IEC-6/CDX2) cells, an absorptive intestinal epithelial cell model.

Glucose-dependent insulinotropic polypeptide regulates dipeptide absorption in mouse jejunum.

Glucose-dependent insulinotropic polypeptide (GIP) secreted from jejunal mucosal K cells augments insulin secretion and plays a critical role in the pathogenesis of obesity and Type 2 diabetes mellitus. In recent studies, we have shown GIP directly activates Na-glucose cotransporter-1 (SGLT1) and enhances glucose absorption in mouse jejunum. It is not known whether GIP would also regulate other intestinal nutrient absorptive processes.

Nucleophosmin, a critical Bax cofactor in ischemia-induced cell death.

We hypothesized that nucleophosmin (NPM), a nucleolar phosphoprotein, is critical for Bax-mediated cell death. To test this hypothesis, Bax activation was induced by metabolic stress. During stress, nucleolar NPM translocated into the cytosol, NPM-Bax complexes formed, and both NPM and Bax accumulated in mitochondria.

Recognition-dependent signaling events in response to apoptotic targets inhibit epithelial cell viability by multiple mechanisms: implications for non-immune tissue homeostasis.

Apoptosis allows for the removal of damaged, aged, and/or excess cells without harm to surrounding tissue. To accomplish this, cells undergoing apoptosis acquire new activities that enable them to modulate the fate and function of nearby cells. We have shown that receptor-mediated recognition of apoptotic versus necrotic target cells by viable kidney proximal tubular epithelial cells (PTEC) modulates the activity of several signaling pathways critically involved in regulation of proliferation and survival.

Role of mitofusin 2 in the renal stress response.

The role of mitofusin 2 (MFN2), a key regulator of mitochondrial morphology and function in the renal stress response is unknown. To assess its role, the MFN2 floxed gene was conditionally deleted in the kidney of mice (MFN2 cKO) by Pax2 promoter driven Cre expression (Pax2Cre). MFN2 cKO caused severe mitochondrial fragmentation in renal epithelial cells that are critical for normal kidney tubular function.

Hexokinase regulates Bax-mediated mitochondrial membrane injury following ischemic stress.

Hexokinase (HK), the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. Since HK isoforms I and II have mitochondrial targeting sequences, we attempted to separate the protective effects of HK on cell metabolism from those on apoptosis. We exposed renal epithelial cells to metabolic stress causing ATP depletion in the absence of glucose and found that this activated glycogen synthase kinase 3β (GSK3β) and Bax caused mitochondrial membrane injury and apoptosis.

Induction of heat shock protein 70 inhibits ischemic renal injury.

Heat shock protein 70 (Hsp70) is a potent antiapoptotic agent. Here, we tested whether it directly regulates renal cell survival and organ function in a model of transient renal ischemia using Hsp70 knockout, heterozygous, and wild-type mice. The kidney cortical Hsp70 content inversely correlated with tubular injury, apoptosis, and organ dysfunction after injury.

Adaptation of intercalated cells along the collecting duct to systemic acid/base changes.

Collecting duct intercalated cells respond to short-term acid/base perturbations by rapidly shuttling H(+)-ATPase to and from the plasma membrane. Purkerson et al. provide information on the regulation of the anion transporters during chronic acidosis and acute recovery (alkalosis).

GSK3beta promotes apoptosis after renal ischemic injury.

The mechanism by which the serine-threonine kinase glycogen synthase kinase-3beta (GSK3beta) affects survival of renal epithelial cells after acute stress is unknown. Using in vitro and in vivo models, we tested the hypothesis that GSK3beta promotes Bax-mediated apoptosis, contributing to tubular injury and organ dysfunction after acute renal ischemia. Exposure of renal epithelial cells to metabolic stress activated GSK3beta, Bax, and caspase 3 and induced apoptosis.

Recognition of apoptotic cells by epithelial cells: conserved versus tissue-specific signaling responses.

During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (m). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence m survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type.

Beta-catenin promotes survival of renal epithelial cells by inhibiting Bax.

Ischemia activates Bax, a proapoptotic BCL2 protein, as well as the prosurvival beta-catenin/Wnt signaling pathway. To test the hypothesis that beta-catenin/Wnt signaling regulates Bax-mediated apoptosis after induction of metabolic stress, which occurs during renal ischemia, we infected immortalized and primary proximal tubular epithelial cells with adenovirus to express either constitutively active or dominant negative beta-catenin constructs. Constitutively active beta-catenin significantly decreased apoptosis and improved cell survival after metabolic stress.

Hsp27 inhibits sublethal, Src-mediated renal epithelial cell injury.

Disruption of cell contact sites in renal epithelial cells contributes to organ dysfunction after ischemia. We hypothesized that heat shock protein 27 (Hsp27), a known cytoprotectant protein, preserves cell architecture and cell contact site function during ischemic stress. To test this hypothesis, renal epithelial cells were subjected to transient ATP depletion, an in vitro model of ischemia-reperfusion injury.

Microscale sample preparation for PCR of C. difficile infected stool.

In this paper, we describe the design of a microfluidic sample preparation chip for human stool samples infected with Clostridium difficile. We established a polymerase chain reaction able to distinguish C. difficile in the presence of several other organisms found in the normal intestinal flora.

Glucose-dependent insulinotropic polypeptide (GIP) stimulates transepithelial glucose transport.

The purpose of this study was to characterize the effects of glucose-dependent insulinotropic peptide (GIP) on small intestinal glucose transport in vitro. Stripped proximal jejunum from fasted mice was mounted in Ussing chambers. The serosal side was bathed in Regular Ringer solution containing 5 mmol/l glucose, and the mucosal side, with solution containing 10 mmol/l 3-O-methyl glucose (3OMG).

Hsp27 inhibits Bax activation and apoptosis via a phosphatidylinositol 3-kinase-dependent mechanism.

Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress.

Vacuolar H+ -ATPase B1 subunit mutations that cause inherited distal renal tubular acidosis affect proton pump assembly and trafficking in inner medullary collecting duct cells.

Point mutations in the B1 subunit of vacuolar H+ -ATPase are associated with impaired ability of the distal nephron to secrete acid (distal renal tubular acidosis). For testing of the hypothesis that these mutations interfere with assembly and trafficking of the H+ -ATPase, constructs that mimic seven known point mutations in inherited distal renal tubular acidosis (M) or wild-type (WT) B1 were transfected into a rat inner medullary collecting duct cell line to express green fluorescence protein (GFP)-B1WT or GFP-B1M fusion proteins. In co-immunoprecipitation studies, GFP-B1WT formed complexes with other H+ -ATPase subunits (c, H, and E), whereas GFP-B1M did not.

Distinct hsp70 domains mediate apoptosis-inducing factor release and nuclear accumulation.

Although hsp70 antagonizes apoptosis-inducing factor (AIF)-mediated cell death, the relative importance of preventing its release from mitochondria versus sequestering leaked AIF in the cytosol remains controversial. To dissect these two protective mechanisms, hsp70 deletion mutants lacking either the chaperone function (hsp70-deltaEEVD) or ATPase function (hsp70-deltaATPase) were selectively overexpressed before exposing cells to a metabolic inhibitor, an insult sufficient to cause mitochondrial AIF release, nuclear AIF accumulation, and apoptosis. Compared with empty vector, overexpression of wild type human hsp70 inhibited bax activation and reduced mitochondrial AIF release after injury.

Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells.

H(+) transport in the collecting duct is regulated by exocytic insertion of H(+)-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain.

Lithium activates the Wnt and phosphatidylinositol 3-kinase Akt signaling pathways to promote cell survival in the absence of soluble survival factors.

Mouse proximal tubular cells (BUMPT), when cultured in the absence of growth factors, activate a default apoptotic pathway. Although Wnt signaling antagonizes the effect of proapoptotic triggers, its role in regulating the default pathway of apoptosis is less well defined. The present study examines the hypothesis that lithium (Li(+)) and (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO), two glycogen synthase kinase-3beta (GSK3beta) inhibitors, promote survival of growth factor-deprived renal epithelial cells by activating the Wnt pathway.

Munc-18-2 regulates exocytosis of H(+)-ATPase in rat inner medullary collecting duct cells.

Exocytic insertion of H(+)-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H(+)-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H(+)-ATPase.

Hsp72 interacts with paxillin and facilitates the reassembly of focal adhesions during recovery from ATP depletion.

The cytoprotective effect of heat stress proteins on epithelial cell detachment, an important cause of acute, ischemic renal failure, was examined after ATP depletion by evaluating focal adhesion complex (FAC) integrity. The intracellular distribution of FAC proteins (paxillin, talin, and vinculin) was assessed by immunohistochemistry before, during, and after exposure of renal epithelial cells to metabolic inhibitors. The resulting ATP depletion caused reversible re-distribution of all three proteins from focal adhesions to the cytosol.

Inhibition of ligand-independent ERK1/2 activity in kidney proximal tubular cells deprived of soluble survival factors up-regulates Akt and prevents apoptosis.

Mouse kidney proximal tubular epithelial (MK-PT) cells die by apoptosis over 7-10 days when deprived of all survival factors. We show here that withdrawal of all survival factors from MK-PT cells is associated with a progressive increase in the activity of extracellular signal-regulated kinase-1 and -2 (ERK1/2) and a progressive decrease in phosphorylated Akt, a kinase critical to cell survival. Pharmacological inhibition of MEK1/2, the immediate upstream kinase for ERK1/2, not only prevented the decrease in phosphorylated Akt, but also prolonged MK-PT cell survival.

HSP72 inhibits apoptosis-inducing factor release in ATP-depleted renal epithelial cells.

Inhibition of the mitochondrial release and nuclear translocation of apoptosis-inducing factor (AIF) by heat stress protein (HSP)72 may ameliorate apoptosis in renal epithelial cells exposed to a metabolic inhibitor. To evaluate this hypothesis, cells were transiently exposed to 5 mM sodium cyanide in the absence of medium glucose, a maneuver known to induce apoptosis. ATP depletion for 1-2 h resulted in the progressive accumulation of mitochondrial AIF in the cytosol of samples obtained by selectively permeabilizing the plasma membrane with digitonin.