Epithelial Transport Research Articles

Dysregulation of intestinal epithelial CFTR-dependent Cl- ion transport and paracellular barrier function drives gastrointestinal symptoms of food-induced anaphylaxis in mice.

Food-triggered anaphylaxis can encompass a variety of systemic and intestinal symptoms. Murine-based and clinical studies have revealed a role for histamine and H1R and H2R-pathway in the systemic response; however, the molecular processes that regulate the gastrointestinal (GI) response are not as well defined. In the present study, by utilizing an IgE-mast cell (MC)-dependent experimental model of oral antigen-induced anaphylaxis, we define the intestinal epithelial response during a food-induced anaphylactic reaction. We show that oral allergen-challenge stimulates a rapid dysregulation of intestinal epithelial transcellular and paracellular transport that was associated with the development of secretory diarrhea. Allergen-challenge induced (1) a rapid intestinal epithelial Cftr-dependent Cl- secretory response and (2) paracellular macromolecular leak that was associated with modification in epithelial intercellular junction proteins claudin-1, 2, 3 and 5, E-cadherin and desmosomal cadherins. OVA-induced Cftr-dependent Cl- secretion and junctional protein degradation was rapid occurring and was sustained for 72 h following allergen-challenge. Blockade of both the proteolytic activity and Cl- secretory response was required to alleviate intestinal symptoms of food-induced anaphylaxis. Collectively, these data suggest that the GI symptom of food-induced anaphylactic reaction, secretory diarrhea, is a consequence of CFTR-dependent Cl- secretion and proteolytic activity.

Gerhard Giebisch (1927–2020): A Leader in Kidney Physiology

Gerhard Giebisch (1927–2020): A Leader in Kidney Physiology

Oral delivery of protein-based therapeutics: Gastroprotective strategies, physiological barriers and in vitro permeability prediction.

The number of biological molecules emerging as therapeutics is growing exponentially due to their higher specificity and tolerability profiles compared to small molecules. Despite this, their traditionally parenteral delivery often results in poor patient compliance and incomplete treatment. Current research is focussed on developing effective oral delivery strategies to facilitate administration of these biomolecules, however no universal method exists to simultaneously provide gastric protection as well as enhance transport across the gastrointestinal epithelium. Furthermore, for efficient formulation development it is imperative that we can reliably analyse permeability of biomolecules through the gastrointestinal tract, highlighting the importance of the continual development and ongoing evaluation of in vitro predictive permeability tools. Here, we review the physiological obstacles associated with peptide and protein delivery throughout the gastrointestinal tract. Furthermore, we highlight methods utilised to circumvent these barriers and promote improved intestinal permeability. Lastly, we explore in vitro models employed to predict epithelial transport. Key findings highlight the need to carefully understand gastrointestinal physiology, allowing specific engineering of oral delivery systems for biomolecules. Significant importance is placed upon understanding enzymatic degradation susceptibility as well as uptake mechanisms for particulate and protein-based therapeutics for the development of successful oral protein delivery platforms.

Phosphorylation of mouse intestinal basolateral amino acid uniporter LAT4 is controlled by food-entrained diurnal rhythm and dietary proteins.

Adaptive regulation of epithelial transporters to nutrient intake is essential to decrease energy costs of their synthesis and maintenance, however such regulation is understudied. Previously we demonstrated that the transport function of the basolateral amino acid uniporter LAT4 (Slc43a2) is increased by dephosphorylation of serine 274 (S274) and nearly abolished by dephosphorylation of serine 297 (S297) when expressed in Xenopus oocytes. Phosphorylation changes in the jejunum of food-entrained mice suggested an increase in LAT4 transport function during food expectation. Thus, we investigated further how phosphorylation, expression and localization of mouse intestinal LAT4 respond to food-entrained diurnal rhythm and dietary protein content. In mice entrained with 18% protein diet, LAT4 mRNA was not submitted to diurnal regulation, unlike mRNAs of luminal symporters and antiporters. Only in duodenum, LAT4 protein expression increased during food intake. Concurrently, S274 phosphorylation was decreased in all three small intestinal segments, whereas S297 phosphorylation was increased only in jejunum. Interestingly, during food intake, S274 phosphorylation was nearly absent in ileum and accompanied by strong phosphorylation of mTORC1 target S6. Entraining mice with 8% protein diet provoked a shift in jejunal LAT4 localization from the cell surface to intracellular stores and increased S274 phosphorylation in both jejunum and ileum during food anticipation, suggesting decreased transport function. In contrast, 40% dietary protein content led to increased LAT4 expression in jejunum and its internalization in ileum. Ex vivo treatments of isolated intestinal villi fraction demonstrated that S274 phosphorylation was stimulated by protein kinase A. Rapamycin-sensitive insulin treatment and amino acids increased S297 phosphorylation, suggesting that the response to food intake might be regulated via the insulin-mTORC1 pathway. Ghrelin, an oscillating orexigenic hormone, did not affect phosphorylation of intestinal LAT4. Overall, we show that phosphorylation, expression and localization of intestinal mouse LAT4 responds to diurnal and dietary stimuli in location-specific manner.

Immunological characterization of two types of ionocytes in the inner ear epithelium of Pacific Chub Mackerel (Scomber japonicus)

The inner ear is essential for maintaining balance and hearing predator and prey in the environment. Each inner ear contains three CaCO3 otolith polycrystals, which are calcified within an alkaline, K+-rich endolymph secreted by the surrounding epithelium. However, the underlying cellular mechanisms are poorly understood, especially in marine fish. Here, we investigated the presence and cellular localization of several ion-transporting proteins within the saccular epithelium of the Pacific Chub Mackerel (Scomber japonicus). Western blotting revealed the presence of Na+/K+-ATPase (NKA), carbonic anhydrase (CA), Na+-K+-2Cl--co-transporter (NKCC), vacuolar-type H+-ATPase (VHA), plasma membrane Ca2+ ATPase (PMCA), and soluble adenylyl cyclase (sAC). Immunohistochemistry analysis identified two distinct ionocytes types in the saccular epithelium: Type-I ionocytes were mitochondrion-rich and abundantly expressed NKA and NKCC in their basolateral membrane, indicating a role in secreting K+ into the endolymph. On the other hand, Type-II ionocytes were enriched in cytoplasmic CA and VHA, suggesting they help transport HCO3- into the endolymph and remove H+. In addition, both types of ionocytes expressed cytoplasmic PMCA, which is likely involved in Ca2+ transport and homeostasis, as well as sAC, an evolutionary conserved acid-base sensing enzyme that regulates epithelial ion transport. Furthermore, CA, VHA, and sAC were also expressed within the capillaries that supply blood to the meshwork area, suggesting additional mechanisms that contribute to otolith calcification. This information improves our knowledge about the cellular mechanisms responsible for endolymph ion regulation and otolith formation, and can help understand responses to environmental stressors such as ocean acidification.

FcRn, but not FcγRs, drives maternal-fetal transplacental transport of human IgG antibodies

The IgG Fc domain has the capacity to interact with diverse types of receptors, including the neonatal Fc receptor (FcRn) and Fcγ receptors (FcγRs), which confer pleiotropic biological activities. Whereas FcRn regulates IgG epithelial transport and recycling, Fc effector activities, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis, are mediated by FcγRs, which upon cross-linking transduce signals that modulate the function of effector leukocytes. Despite the well-defined and nonoverlapping functional properties of FcRn and FcγRs, recent studies have suggested that FcγRs mediate transplacental IgG transport, as certain Fc glycoforms were reported to be enriched in fetal circulation. To determine the contribution of FcγRs and FcRn to the maternal-fetal transport of IgG, we characterized the IgG Fc glycosylation in paired maternal-fetal samples from patient cohorts from Uganda and Nicaragua. No differences in IgG1 Fc glycan profiles and minimal differences in IgG2 Fc glycans were noted, whereas the presence or absence of galactose on the Fc glycan of IgG1 did not alter FcγRIIIa or FcRn binding, half-life, or their ability to deplete target cells in FcγR/FcRn humanized mice. Modeling maternal-fetal transport in FcγR/FcRn humanized mice confirmed that only FcRn contributed to transplacental transport of IgG; IgG selectively enhanced for FcRn binding resulted in enhanced accumulation of maternal antibody in the fetus. In contrast, enhancing FcγRIIIa binding did not result in enhanced maternal-fetal transport. These results argue against a role for FcγRs in IgG transplacental transport, suggesting Fc engineering of maternally administered antibody to enhance only FcRn binding as a means to improve maternal-fetal transport of IgG.

The Role of TREK-1 and AQP5 in Gonadocorticoid-Related Voice Disorders

TWIK-related potassium channel-1 (TREK-1) and Aquaporin 5 (AQP5) are involved in epithelial integrity and fluid transport, respectively. In this study, we aimed to compare physiological and gestational patterns of TREK-1 and AQP5 location and expression in rat larynx. Our secondary objective was to reveal the effect of estradiol (E2) and progesterone (PG) on these two biomolecules. This study was conducted on 20 Wister albino female rats which were assigned as control (group A) and pregnant group (group B). The rats were sacrificed at 20th day of pregnancy. Blood was obtained directly from the ventricle for detection of serum E2 and PG levels. Larynx was resected for immunohistochemical analyses and real-time polymerase chain reaction testing for detection of TREK-1 and AQP5 staining and expression, respectively. Relative TREK-1 (P=0.035) and AQP5 (P=0.019) expression was found to be significantly high in group B when compared with group A. We found positive correlation between serum E2 levels and both biomolecules (TREK-1; P=0.018, AQP5; P=0.016). We also found positive correlation between serum PG levels and both biomolecules (TREK-1; P=0.001, AQP5; P=0.019). TREK-1 immunostaining was found to be higher in surface epithelium and lamina propria of vocal cord mucosa. AQP5 was particularly found to be located in basement membrane and adjacent superficial lamina propria. We revealed the physiological and gestational pattern of laryngeal TREK-1 and AQP5 expression for the first time. Gestational expression of both TREK-1 and AQP5 was found to be increased. Stimulatory effect of E2 and PG on laryngeal TREK-1 and AQP5 expression was also revealed. We revealed upregulatory effect of E2 and PG on laryngeal TREK-1 and AQP5 expression. Based on this finding, it can be suggested that TREK-1 and AQP5 play role in biomolecular processes leading gonadocorticoid-related voice changes.

Co-cultures of renal progenitors and endothelial cells on kidney decellularized matrices replicate the renal tubular environment in vitro.

Herein we propose creating a bilayer tubular kidney in-vitro model. It is hypothesized that membranes composed of decellularized porcine kidney extracellular matrix are valid substitutes of the tubular basement membrane by mimicking the physiological relevance of the in vivo environment and disease phenotypes. Extracellular matrix was obtained from decellularized porcine kidneys. After processing by lyophilization and milling, it was dissolved in an organic solvent and blended with poly(caprolactone). Porous membranes were obtained by electrospinning and seeded with human primary renal progenitor cells to evaluate phenotypic alterations. To create a bilayer model of the in vivo tubule, the same cells were differentiated into epithelial tubular cells and co-cultured with endothelial cells in opposite sites. Our results demonstrate increasing metabolic activity, proliferation and total protein content of renal progenitors over time. We confirmed the expression of several genes encoding epithelial transport proteins and we could also detect tubular-specific proteins by immunofluorescence stainings. Functional and transport assays were performed trough the bilayer by quantifying both human serum albumin uptake and inulin leakage. Furthermore, we validated the chemical modulation of nephrotoxicity on this epithelium-endothelium model by cisplatin exposure. The use of decellularized matrices in combination with primary renal cells was shown to be a valuable tool for modelling renal function and disease in vitro. We successfully validated our hypothesis by replicating the physiological conditions of an in vitro tubular bilayer model. The developed system may contribute significantly for the future investigation of advanced therapies for kidney diseases.

Decreased airway epithelial ion transport was associated with the severity of the respiratory syncytial virus infection and complications in infants.

Decreased airway epithelial ion transport was associated with the severity of the respiratory syncytial virus infection and complications in infants.

Functional genomics analysis of human colon organoids identifies key transcription factors.

Organoids are a valuable three-dimensional (3D) model to study the differentiated functions of the human intestinal epithelium. They are a particularly powerful tool to measure epithelial transport processes in health and disease. Though biological assays such as organoid swelling and intraluminal pH measurements are well established, their underlying functional genomics are not well characterized. Here we combine genome-wide analysis of open chromatin by ATAC-Seq with transcriptome mapping by RNA-Seq to define the genomic signature of human intestinal organoids (HIOs). These data provide an important tool for investigating key physiological and biochemical processes in the intestinal epithelium. We next compared the transcriptome and open chromatin profiles of HIOs with equivalent data sets from the Caco2 colorectal carcinoma line, which is an important two-dimensional (2D) model of the intestinal epithelium. Our results define common features of the intestinal epithelium in HIO and Caco2 and further illustrate the cancer-associated program of the cell line. Generation of Caco2 cysts enabled interrogation of the molecular divergence of the 2D and 3D cultures. Overrepresented motif analysis of open chromatin peaks identified caudal type homeobox 2 (CDX2) as a key activating transcription factor in HIO, but not in monolayer cultures of Caco2. However, the CDX2 motif becomes overrepresented in open chromatin from Caco2 cysts, reinforcing the importance of this factor in intestinal epithelial differentiation and function. Intersection of the HIO and Caco2 transcriptomes further showed functional overlap in pathways of ion transport and tight junction integrity, among others. These data contribute to understanding human intestinal organoid biology.

Epithelial transport in digestive diseases: mice, monolayers, and mechanisms.

The transport of electrolytes and fluid by the intestinal epithelium is critical in health to maintain appropriate levels of fluidity of the intestinal contents. The transport mechanisms that underlie this physiological process are also subject to derangement in various digestive disease states, such as diarrheal illnesses. This article summarizes the 2019 Hans Ussing Lecture of the Epithelial Transport Group of the American Physiological Society and discusses some pathways by which intestinal transport is dysregulated, particularly in the setting of infection with the diarrheal pathogen, Salmonella, and in patients treated with small-molecule inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFr-TKI). The burdensome diarrhea in patients infected with Salmonella may be attributable to decreased expression of the chloride-bicarbonate exchanger downregulated in adenoma (DRA) that participates in electroneutral NaCl absorption. This outcome is possibly secondary to increased epithelial proliferation and/or decreased epithelial differentiation that occurs following infection. Conversely, the diarrheal side effects of cancer treatment with EGFr-TKI may be related to the known ability of EGFr-associated signaling to reduce calcium-dependent chloride secretion. Overall, the findings described may suggest targets for therapeutic intervention in a variety of diarrheal disease states.

Novel Human Polymorphisms Define a Key Role for the SLC26A6-STAS Domain in Protection From Ca2+-Oxalate Lithogenesis.

Impaired homeostasis of the carboxylic acids oxalate and citrate, dramatically increases the risk for the formation of Ca2+-oxalate kidney stones, which is the most common form of kidney stones in humans. Renal homeostasis of oxalate and citrate is controlled by complex mechanisms including epithelial transport proteins such as the oxalate transporter, SLC26A6, and the citrate transporters, the SLC13’s. These transporters interact via the SLC26A6-STAS domain in vitro, however, the role of the Sulfate Transporter and Anti-Sigma factor antagonist (STAS) domain in Ca2+-oxalate stone formation was not investigated in humans. Here, we report two novel human SLC26A6 polymorphisms identified in the STAS domain of SLC26A6 in two heterozygous carriers. Intriguingly, these individuals have low urinary citrate, but different clinical manifestations. Our in vitro experiments indicate that the homolog mutations of SLC26A6(D23H/D673N) and SLC26A6(D673N) alone abolished the expression and function of SLC26A6, and impaired the regulation of SLC13-mediated citrate transport by SLC26A6. On the other hand, the SLC26A6(R621G) variant showed reduced SLC26A6 protein expression and membrane trafficking, retained full transport activity, but impaired the regulation of the citrate transporter. Accordingly, the human SLC26A6(D23H/D673N) carrier showed a dramatic reduction in urinary citrate concentrations which resulted in Ca2+-oxalate stones formation, as opposed to the carrier of SLC26A6(R621G). Our findings indicate that the human SLC26A6-STAS domain mutations differentially impair SLC26A6 expression, function, and regulation of citrate transporters. This interferes with citrate and oxalate homeostasis thus potentially predisposes to Ca2+-oxalate kidney stones.

A Flexible and Reproducible Framework for Modelling Renal Tubules

We have developed a new framework for defining renal tubule models utilising a repository of existing models from the literature. The CellML format ( https://cellml.org) is an XML format for encoding mathematical models ‐ typically used for lumped parameter, non‐spatial models of physiological systems. We have previously demonstrated how models of epithelial transport processes can be discovered in public model repositories such as the Physiome Model Repository ( https://models.physiomeproject.org) and reused in building novel models at the epithelial cellular spatial scale.In this work we present a novel Python‐based framework in which we extend our previous work to now look at solute transport and flow in generic tubule segments. The Python code captures the standard equations for flow and solute transport and we link to CellML descriptions of transport mechanisms to define the transport of solutes between the lumen, intracellular space, and interstitial fluid.By linking to models of, for example, epithelial transport proteins encoded in the CellML format we are able to configure the generic tubule segment to represent known physiology of the various nephron segments in the kidney. As the number and variety of suitable models in the model repository grows, we will be able to “automatically” discover and utilize them in this framework.In future work, we will are attempting to utilize clinical data captured in semantic knowledge frameworks, such as openEHR, to guide the discovery and parameterization of the epithelial cellular transport processes based on clinical or experimental observations.Support or Funding InformationWe gratefully acknowledge funding from the Aotearoa Foundation, Medical Technologies Centre of Research Excellence, and the Auckland Bioengineering Institute.

Epithelial Mechanosensitive Ion Channel Piezo2 Contributes to Pressure‐Induced Epithelial Chloride Secretion in Mouse Colon

BACKGROUNDMechanical stimulation of the gastrointestinal (GI) epithelium causes changes in epithelial ion transport by an unclear mechanism. We recently discovered the mechanosensitive ion channel Piezo2 is specifically expressed by the enteroendocrine cells (EECs) and is necessary for EEC mechanosensitivity. Further, in mouse small bowel, the pharmacologic inhibition or genetic knockout of the epithelial Piezo2 led to an inhibition of the pressure‐induced short‐circuit current (Isc) in the Ussing chamber. These results suggested that EEC Piezo2 plays a role in pressure‐induced epithelial ion transport, but the mechanism and whether these responses are region‐specific remains uknown.AIMDetermine whether epithelial Piezo2‐mediated pressure‐induced Isc is due to Clsecretion in the mouse colon.METHODSFull‐thickness mid‐colon preparations of VilCre/+::Piezo2f/f (Piezo2CKO) or Vil+/+::Piezo2f/f (Piezo2WT) mice at 0.3 cm2 were mounted in 4mL Ussing chambers with normal Krebs solution. The epithelial side was intermittently pressure‐clamped at a range of pressures (5, 10, 20, 30, 40, 60 mmHg) for 10s each. Forskolin (FSK, 10μM) was applied at the end of the experiments, and FSK response was used to normalize the pressure‐induced responses (ΔIsc/ΔIsc,FSK).RESULTSPressure applied to the mucosa of Piezo2CKO and Piezo2WTcolons caused a pressure‐dependent increase in Isc. Responses in Piezo2CKO colons were smaller than Piezo2WT at 40mmHg (0.41±0.1 vs. 0.76±0.1) and 60mmHg (0.51±0.1 vs. 0.87±0.1) (n=9–11, P<0.05, two‐way ANOVA). To determine the nature of the pressure‐induced Isc we removed Cl− from the Krebs solutions. Cl‐removal resulted in XX. We found a nearloss in the mechanically induced ΔIsc in both Piezo2WT tissues and Piezo2CKO: 5mmHg (− 88.85±0.1% and −87.44±0.1% Piezo2WT), 10mmHg (−86.46±0.1% Piezo2CKO and − 90.30±0.1% Piezo2WT), 20mmHg (−88.50±0.01% Piezo2CKO and −89.86±0.1% Piezo2WT), 40mmHg (−91.78±0.1% Piezo2CKO and −91.35±0.04% Piezo2WT), and 60mmHg (−93.93±0.04% Piezo2CKO and −92.08±0.04% Piezo2WT (n= 5–10, P <0.05, unpaired t‐test). Replacement of Cl− led to full recovery of the mechanically induced ΔIsc in both Piezo2WT and Piezo2CKO tissues when tested at 30 mmHg (Piezo2WT: 2.51±1.4 vs. 73.52±33.8, n=6 and 3, P <0.05, unpaired t‐test), (Piezo2CKO: 1.80±1.0 vs. 45.14±12.4, n=5 and 4, P <0.05, unpaired t‐test).CONCLUSIONSHydrostatic pressure applied to the colon epithelium leads to an increase in epithelial secretion that depends chloride and Piezo2 contributes to this mechanosensitive chloride secretion.Support or Funding InformationSupported by AGA RSA, NIH DK106456, NIH DK119683, NIH DK123549

Editorial: Biographical Memoirs , Volume 68

Editorial: <i>Biographical Memoirs</i> , Volume 68

Heterogeneous distribution of ion transporters in enteroids enriched in different intestinal epithelial cell types

The small intestine which is characterized by different cell types lining specific sites along the crypt‐villus axis plays a critical role in water, electrolyte and mineral balance. Normally, net fluid absorption by the small intestine occurs mainly because ion‐dependent fluid absorption in the villus column is greater than ion‐driven fluid secretion in the crypt. To generate these site‐specific ion gradients, appropriate transporters must be strategically located in cells lining the crypt or villus regions. The multivalent ions calcium and iron also function as essential minerals, and deficiency in these minerals is highly prevalent worldwide. The crypt‐villus site of many electrolyte and mineral transporters has been localized by immunocytochemistry, but their expression in rare cell types like stem cells (ISC) is unknown. To test the hypothesis that representative ion and mineral transporters are distributed heterogeneously among the various intestinal cell types, we made enteroids enriched in ISC, enterocytes (ENT), goblet (GOB), and Paneth (PAN) cells. We then confirmed by cell‐type specific biomarkers that ISC, ENT, GOB and PAN enteroids were enriched in stem cells (~90% of all cells per organoid), enterocytes (90%), goblet (80%) and Paneth (65%) cells, respectively. ENT and GOB enteroids had the highest mRNA levels of the chloride‐anion exchanger DRA and sodium‐hydrogen exchanger NHE3, while the sodium‐bicarbonate cotransporter NBCe1 was expressed primarily in ENT enteroids. Conversely, the chloride channel CFTR and sodium‐potassium‐chloride cotransporter NKCC1 mRNA were mainly expressed in ISC and PAN enteroids. Findings confirm previous immunocytochemical localization of NBCe1 and DRA in the villus region lined by enterocytes, with CFTR and NKCC1 mainly in intestinal crypts containing Paneth and stem cells. Basolateral NKCC1 staining was observed in ISC and PAN, but not in ENT and GOB, enteroids. The membrane location and expression of NKCC1 in crypt‐dwelling cells reflect its prominent role in intestinal fluid secretion. Members of the intestinal epithelial transport system for iron (DMT1, FTL, IREG1 and HEPH) showed homogeneous distribution in all cell types. The calcium transporters TRPV6 and PMCA1 were expressed least in ENT and GOB, respectively. Our findings depict a mostly heterogeneous distribution of ion transporters in different cell types, reflecting their likely distribution along the crypt villus axis in vivo, suggesting that appropriate expression in different cell types may be essential for ion and mineral homeostasis.Support or Funding InformationSupported by NSF 1456673, 1754783 (RF) and NIH R01AT010243 (RF, NG)

Analysis of Protein Phosphatase 1 (PP1) and SPAK/OSR1 as KCC3a Phosphorylation/Dephosphorylation Cycle Regulators

Study ObjectiveTo identify the phosphatase/kinase complex that regulates KCC3a activation/deactivation during cell volume regulation and ion transport.BackgroundThe K‐Cl cotransporters (KCCs) are SLC12A cation/chloride cotransporters (CCCs) that mediate epithelial transport, maintain cellular volume, and regulate GABA neurotransmission. These mechanisms are complex, and contain multiple sensors, transducers, and effectors, often operating in parallel pathways. It has been shown that direct phosphorylation by SPAK/OSR1 kinases regulate KCCs activities. In particular for KCC3a phosphorylation of regulatory site Thr1039 by these kinases results in inhibited K‐Cl cotransport, whereas dephosphorylation of this residue greatly stimulates KCC3 activity. Protein phosphatases (PP) inhibitors reduce KCCs function and so, it is generally accepted the necessary presence of phosphatases in the WNKs/SPAK/CCCs/signaling pathway. Several studies have proposed protein phosphatase 1 as the major PP responsible for KCCs dephosphorylation. Specificity of PP1 catalytic domain is determined by its association with one or two regulatory subunits that modulate its activity. In the present study we analyzed how exactly is PP1 regulating KCCs activities and the possible role of SPAK/OSR1 kinases as PP1 regulatory subunits.MethodsHEK 293 cells and Xenopus laevis oocytes were either transfected or microinjected with KCC3 and wild type (WT) or mutant PP1 plasmids (H248K or T320A). KCC3 dephosphorylation in response to cell swelling, hypotonic conditions or presence of “active/inactive” PP1 in dephosphorylation assays, was evaluated by Western blot analysis of KCCs regulatory phosphorylation sites (pThr1039 and pThr991 in KCC3a). Phosphorylation of SPAK/OSR1 activating regulatory site Thr273 was analyzed in parallel under the same conditions. Formation of a phosphatase/kinase/cotransporter complex was evaluated by immunoprecipitation assays. Dephosphorylation assays were performed either with immunoprecipitated PP1 from cell extracts or with recombinant PP1.ResultsWT or constitutive active mutant PP1 T320A induced KCC3a Thr1039 dephosphorylation in HEK cells and X. laevis oocytes even under isotonic conditions, where KCC3a is normally phosphorylated. PP1 inactivating mutation H248K prevented KCC3 dephosphorylation in response to hypotonic conditions and greatly promoted Thr1039 phosphorylation. Dephosphorylation assays in presence of “active” or “inactive” immunoprecipitated WT PP1, showed that “active” PP1 promotes SPAK Thr273 dephosphorylation, while “inactive” PP1 greatly increased SPAK phosphorylation and thereby KCC3 T1039 inmunoprecipitated from hypotonic lysates where both proteins are found dephosphorylated.ConclusionsOur results show a direct role for PP1 in KCC3 function. PP1 is regulating KCC3 activity via SPAK/OSR1 kinases. We show “active” and “inactive” PP1 regulates SPAK/OSR1 activity, that in turn will directly affect KCCs cell volume regulatory response.Support or Funding InformationConacyt 283555Papiit IA207718

Development of a microemulsion for encapsulation and delivery of gallic acid. The role of chitosan

A novel water-in-oil (W/O) microemulsion based on natural oils, namely extra virgin olive oil (EVOO) and sunflower oil (SO), in the presence of non-ionic surfactants was successfully formulated. The novel microemulsion was used as a carrier for gallic acid (GA) to assure its protection and efficacy upon nasal administration. The work presents evidence that this microemulsion can be used as a nasal formulation for the delivery of polar antioxidants, especially, after incorporation of chitosan (CH) in its aqueous phase. The structure of the system was studied by Small Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS) and Electron Paramagnetic Resonance (EPR) spectroscopy techniques. By the addition of CH, the diameter of the microemulsion remained unaltered at 47 nm whereas after the incorporation of GA, micelles with 51 nm diameter were detected. The dynamic properties of the surfactant monolayer were affected by both the incorporation of CH and GA. Moreover, the antioxidant activity of the latter remained unaltered (99 %). RPMI 2650 cell line was used as the in vitro model for cell viability and for GA nasal epithelial transport studies after microemulsion administration. The results suggested that the nasal epithelial permeation of GA was enhanced, 3 h post administration, by the presence of 0.2 % v/v microemulsion in the culture medium. However, the concentration of the transported antioxidant in the presence of CH was higher indicating the polymer’s effect on the transport of the GA. The study revealed that nasal administration of hydrophilic antioxidants could be used as an alternative route besides oral administration.

WNK1 Kinase Stimulates Angiogenesis to Promote Tumor Growth and Metastasis.

With-no-lysine (K)-1 (WNK1) is the founding member of family of four protein kinases with atypical placement of catalytic lysine that play important roles in regulating epithelial ion transport. Gain-of-function mutations of WNK1 and WNK4 cause a mendelian hypertension and hyperkalemic disease. WNK1 is ubiquitously expressed and essential for embryonic angiogenesis in mice. Increasing evidence indicates the role of WNK kinases in tumorigenesis at least partly by stimulating tumor cell proliferation. Here, we show that human hepatoma cells xenotransplanted into zebrafish produced high levels of vascular endothelial growth factor (VEGF) and WNK1, and induced expression of zebrafish wnk1. Knockdown of wnk1 in zebrafish decreased tumor-induced ectopic vessel formation and inhibited tumor proliferation. Inhibition of WNK1 or its downstream kinases OSR1 (oxidative stress responsive kinase 1)/SPAK (Ste20-related proline alanine rich kinase) using chemical inhibitors decreased ectopic vessel formation as well as proliferation of xenotransplanted hepatoma cells. The effect of WNK and OSR1 inhibitors is greater than that achieved by inhibitor of VEGF signaling cascade. These inhibitors also effectively inhibited tumorigenesis in two separate transgenic zebrafish models of intestinal and hepatocellular carcinomas. Endothelial-specific overexpression of wnk1 enhanced tumorigenesis in transgenic carcinogenic fish, supporting endothelial cell-autonomous effect of WNK1 in tumor promotion. Thus, WNK1 can promote tumorigenesis by multiple effects that include stimulating tumor angiogenesis. Inhibition of WNK1 may be a potent anti-cancer therapy.

The different expression of glycogen phosphorylases in renal clear cell renal carcinoma and chromophobe renal carcinoma

BackgroundThe various pathogenesis between Clear cell renal carcinoma (CCRCC) and Chromophobe renal carcinoma (CHRCC) contributes to the different tumor growth rate and metastasis. In this study, we explored the distinct proteomic profiles between these two cancers and found different expression of glycogen phosphorylases in two cancers.MethodsWe explored novel targets by proteomics. Five CCRCC cases and five CHRCC cases were selected for tandem mass tag-labeling liquid chromatography-mass spectroscopy (LC–MS). Gene ontology and KEGG pathway were applied for bioinformatic analysis. Glycogen phosphorylases were detected by Western blotting.ResultsCHRCC were younger, more commonly female, and had larger tumors compared to those with CCRCC. 101 differentially expressed proteins (DEPs) in CCRCC and 235 DEPs in CHRCC were detected by LC–MS. It was found that disruption of metabolic pathways, epithelial cell differentiation, and cell response were the common characters for two tumor types. Activation of cell–cell adhesion and oxidation–reduction process stimulate CCRCC growth and epithelial cell differentiation and transferrin transport was involved in CHRCC growth, We also found that oxidative phosphorylation is activated in CHRCC and inhibited in CCRCC. More importantly, we found and confirmed that upregulation of glycogen phosphorylase liver type in CCRCC and glycogen phosphorylase brain type in CHRCC mediated differential glycogenolysis in the two tumor types, which could serve as potential therapeutic targets.ConclusionWe found different expression of glycogen phosphorylases in CCRCC and CHRCC by quantitative proteomics, which provides potential therapeutic targets in the future.