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Cited by 2Ion Transport and Channel RegulationDrug Transport and Resistance MechanismsDigestive system and related health

Regulation of Enterocyte Brush Border Membrane Primary Na-Absorptive Transporters in Human Intestinal Organoid-Derived Monolayers

Open access
Haynes, Jennifer, Palaniappan, Balasubramanian, Crutchley, John M., Sundaram, Uma·2024·Cells
DOI 10.3390/cells13191623PMC PMC11482628
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Abstract

In the small intestine, sodium (Na) absorption occurs primarily via two apical transporters, Na-hydrogen exchanger 3 (NHE3) and Na-glucose cotransporter 1 (SGLT1). The two primary Na-absorptive pathways were previously shown to compensatorily regulate each other in rabbit and rat intestinal epithelial cells. However, whether NHE3 and SGLT1 regulate one another in normal human enterocytes is unknown, mainly due to a lack of appropriate experimental models. To investigate this, we generated 2D enterocyte monolayers from human jejunal 3D organoids and used small interfering RNAs (siRNAs) to knock down NHE3 or SGLT1. Molecular and uptake studies were performed to determine the effects on NHE3 and SGLT1 expression and activity. Knockdown of NHE3 by siRNA in enterocyte monolayers was verified by qPCR and Western blot analysis and resulted in reduced NHE3 activity. However, in NHE3 siRNA-transfected cells, SGLT1 activity was significantly increased. siRNA knockdown of SGLT1 was confirmed by qPCR and Western blot analysis and resulted in reduced SGLT1 activity. However, in SGLT1 siRNA-transfected cells, NHE3 activity was significantly increased. These results demonstrate for the first time the functionality of siRNA in patient-derived organoid monolayers. Furthermore, they show that the two primary Na absorptive pathways in human enterocytes reciprocally regulate one another.

AI snapshot

Written from the paper’s full text.

TL;DR

Using siRNA in human jejunal organoid-derived enterocyte monolayers, knockdown of NHE3 increased SGLT1 activity and knockdown of SGLT1 increased NHE3 activity, demonstrating reciprocal regulation of the two primary apical Na-absorptive transporters in normal human enterocytes. The study also provides first proof-of-concept that siRNA-mediated gene silencing works in patient-derived intestinal organoid monolayers.

Key findings

  • NHE3 siRNA knockdown was verified by qPCR and Western blot and significantly reduced NHE3 activity, measured as Na-dependent, EIPA-sensitive 22Na uptake; however, SGLT1 activity was significantly increased, measured as Na-dependent, phlorizin-sensitive 3H-OMG uptake.
  • SGLT1 siRNA knockdown was verified by qPCR and Western blot and significantly reduced SGLT1 activity; however, NHE3 activity was significantly increased.
  • The authors conclude that NHE3 and SGLT1 reciprocally regulate one another in normal human enterocytes, consistent with prior compensatory regulation seen in mammalian intestinal cell models.
  • Differentiation of jejunal monolayers was supported by decreased LGR5 and MKI67 and increased FABP2 and NHE3 expression, plus ZO-1 immunostaining and alkaline phosphatase activity.

Methods

  • Human jejunal 3D organoids from a healthy donor (organoid model MU-007) were differentiated into 2D enterocyte monolayers; experiments used confluent monolayers on day 5 or 6.
  • siRNA (15 nM) targeting human NHE3 (SLC9A3), human SGLT1 (SLC5A1), or non-targeting negative control was transfected with Lipofectamine RNAiMAX one day after plating; knockdown was assessed by qPCR and Western blot.
  • Transport activity was measured by radiotracer uptake: NHE3 as Na-dependent, EIPA-sensitive 22Na uptake and SGLT1 as Na-dependent, phlorizin-sensitive 3H-OMG uptake. Data are mean ± SEM, analyzed by two-tailed Student’s t test with significance at p < 0.05.

Limitations

  • The paper does not include a dedicated limitations section; it notes that siRNA is transient and that siRNA use in organoid-derived monolayers had not been previously reported, but it does not discuss limitations of the findings.
  • Evident from the text: experiments used a single healthy donor jejunal organoid line (MU-007), so donor variability, other intestinal segments, and disease states are not addressed.
  • Evident from the text: no rescue experiments, dose-response, mechanism/signaling pathway dissection, or off-target controls beyond non-targeting siRNA are reported; exact effect sizes are not given, only statistical significance.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/cells13191623

Cites

66 of 66 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

36 of 55 cited sources checked · 23 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    NHE3 and SGLT1 have been shown to regulate one another in mammalian intestinal epithelial cell models, including rabbit and rat cells.

    “The two primary Na-absorptive pathways were previously shown to compensatorily regulate each other in rabbit and rat intestinal epithelial cells.”
    • Also stated in: § 1. Introduction · plain
      “In a separate study, NHE3 and SGLT1 were shown to reciprocally regulate one another in vitro in rat intestinal epithelial cells [27].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [22] Lehmann, 2016, Intestinal SGLT1 in metabolic health and disease, Am. J. Physiol. Liver Physiol. source

      The “GI Motility and Secretion Associated With SGLT1 Inhibition” section (page not provided) reports reciprocal regulation of SGLT1 and NHE3 in cell cultures: suppressing either enhances the other. The References identify the cited cell study as involving rat intestinal epithelial cells, but our copy does not establish the same compensatory regulation in rabbit cells. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [27] Coon, 2011, Reciprocal regulation of the primary sodium absorptive pathways in rat intestinal epithelial cells, Am. J. Physiol. Physiol. source

      The Results and Discussion report reciprocal, compensatory regulation of NHE3 and SGLT1 in rat IEC-18 intestinal epithelial cells. The Discussion mentions rabbit small-intestine studies only as suggesting an indirect link between the pathways, not as showing the reciprocal regulation claimed for rabbit cells. The rabbit portion is therefore unsupported by this source. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation

    Immortalized colon cancer cell lines have demonstrated limited clinical translatability.

    “It is therefore not clear if these models accurately reflect normal expression, function, and regulation of intestinal epithelial cell transporters, but so far, immortalized colon cancer cells lines have demonstrated limited clinical translatability [41,42].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [41] Faber, S.C., Lahoti, T.S., Taylor, E.R., Lewis, L., Sapiro, J.M., Sales, V.T., Dragan, Y.P., and Jeffy, B.D. (2022). Current Therapeutic Landscape and Safety Roadmap for Targeting the Aryl Hydrocarbon Receptor in Inflammatory Gastrointestinal Indications. Cells, 11. source
    • cites [42] Yamaura, 2015, Functional Comparison of Human Colonic Carcinoma Cell Lines and Primary Small Intestinal Epithelial Cells for Investigations of Intestinal Drug Permeability and First-Pass Metabolism, Drug Metab. Dispos. source

      The Discussion reports that LS180 and Caco-2 cells are poorly suited to assessing intestinal permeability and first-pass CYP3A4 metabolism simultaneously, and the Abstract says none of the tested cell lines fully replicated small-intestinal drug-disposition properties. That supports limitations of these models, but not a general finding of limited clinical translatability: the paper does not test clinical predictive performance, and its Introduction says Caco-2 permeability correlates well with drug absorption in humans. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Findingown data

    Compared to undifferentiated organoids, expression of the intestinal stem cell marker LGR5 and the proliferation marker MKI67 was substantially decreased in differentiated monolayers.

    “Compared to undifferentiated organoids, expression of the intestinal stem cell marker LGR5 and the proliferation marker MKI67 was substantially decreased in differentiated monolayers (Figure 1C), in accordance with an earlier study performed in human duodenal organoid-derived monolayers [16].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [16] Yin, 2018, Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers, Cell. Mol. Gastroenterol. Hepatol. source
  • Premisecitation

    In the human small intestine, Na absorption occurs primarily via two apical transporters, NHE3 and SGLT1.

    “In the small intestine, sodium (Na) absorption occurs primarily via two apical transporters, Na-hydrogen exchanger 3 (NHE3) and Na-glucose cotransporter 1 (SGLT1).”
    • Also stated in: § 1. Introduction · plain
      “The two main pathways of Na absorption in the human small intestine are BBM transporters Na-hydrogen exchanger 3 (NHE3; SLC9A3) and Na-glucose cotransporter 1 (SGLT1; SLC5A1).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [19] Kiela, 2016, Physiology of Intestinal Absorption and Secretion, Best Pr. Res. Clin. Gastroenterol. source

      The ‘Intestinal Na+ absorption’ section identifies NHE3 as the most significant apical Na+/H+ exchanger, and the ‘Sugar transport across the GI tract’ section describes SGLT1-mediated Na+ uptake with glucose. But the paper also describes Na+ absorption through other nutrient transporters, including amino-acid transporters; it does not establish that NHE3 and SGLT1 together account for most small-intestinal Na+ absorption. Page numbers were not available in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    The reciprocal regulation between NHE3 and SGLT1 in human enterocytes is consistent with compensatory regulation observed in mammalian intestinal cell models.

    “This is consistent with the compensatory regulation between NHE3 and SGLT1 observed in mammalian intestinal cell models [24,25,27], and therefore serves as proof of concept for using siRNA technology in patient-derived organoid monolayers.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [24] Palaniappan, 2018, Direct and specific inhibition of constitutive nitric oxide synthase uniquely regulates brush border membrane Na-absorptive pathways in intestinal epithelial cells, Nitric Oxide source
    • cites [25] Palaniappan, 2019, Stimulation of constitutive nitric oxide uniquely and compensatorily regulates intestinal epithelial cell brush border membrane Na absorption, Physiol. Rep. source
    • cites [27] Coon, 2011, Reciprocal regulation of the primary sodium absorptive pathways in rat intestinal epithelial cells, Am. J. Physiol. Physiol. source
  • Classified as probably not a substantial claim. 14
  • May combine two claims. 13

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Patient-derived intestinal 3D organoid culture models and their increasing use over the last decade have greatly facilitated investigation of human intestinal physiology and regulation.

    “The establishment of patient-derived intestinal 3D organoid culture models [9] and their increasing use over the last decade has greatly facilitated the investigation of human intestinal physiology and regulation [43].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [9] Sato, 2011, Long-term Expansion of Epithelial Organoids from Human Colon, Adenoma, Adenocarcinoma, and Barrett’s Epithelium, Gastroenterology source
    • cites [43] Winner, 2022, Organoids in gastrointestinal diseases: From experimental models to clinical translation, Gut source

  • May combine two claims.

    Premisecitation

    Enhanced intracellular cNO inhibits NHE3 but stimulates SGLT1 in differentiated human jejunal organoid-derived monolayers.

    “Consistent with our previous studies performed in mammalian intestinal epithelial cell models [25], we recently showed that enhanced intracellular cNO inhibits NHE3 but stimulates SGLT1 in differentiated human jejunal organoid-derived monolayers [18].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [18] Haynes, 2022, Regulation of nutrient and electrolyte absorption in human organoid-derived intestinal epithelial cell monolayers, Transl. Res. source
    • cites [25] Palaniappan, 2019, Stimulation of constitutive nitric oxide uniquely and compensatorily regulates intestinal epithelial cell brush border membrane Na absorption, Physiol. Rep. source

      The Discussion reports that increased intracellular cNO inhibits NHE3 and stimulates SGLT1. But it identifies the studied cells as coming from rabbits and rats, in vivo and in vitro, respectively—not differentiated human jejunal organoid-derived monolayers. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Immortalized colon cancer cell lines such as Caco-2, HT-29, and T84 contain vastly mutated genomes and altered transcriptional and metabolic profiles.

    “Until recently, human cell culture models for studying intestinal epithelial cell transport have generally used colon cancer cell lines such as Caco-2, HT-29, and T84 [38,39], which are immortalized and contain vastly mutated genomes in addition to altered transcriptional and metabolic profiles [40].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [40] In, 2016, Human mini-guts: New insights into intestinal physiology and host–pathogen interactions, Nat. Rev. Gastroenterol. Hepatol. source

      The “Limitations of traditional intestinal models” section identifies Caco-2, HT29 and T84 as immortalized, cancer-derived cell lines and reports differences in protein expression from the human intestine. It does not say that their genomes are vastly mutated or that they have altered metabolic profiles; its protein-expression findings do not establish the broader claim about transcriptional profiles. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    NHE3 exchanges one luminal Na ion for one intracellular H ion and is the principal NHE isoform expressed in the small intestinal brush border membrane.

    “NHE3 exchanges one luminal Na ion for one intracellular H ion and is the principal NHE isoform expressed in the small intestinal BBM [20].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [20] Nikolovska, 2022, The Role of Plasma Membrane Sodium/Hydrogen Exchangers in Gastrointestinal Functions: Proliferation and Differentiation, Fluid/Electrolyte Transport and Barrier Integrity, Front. Physiol. source

      The small-intestine crypt–villus diagram caption identifies NHE3 as the major brush-border NHE isoform responsible for Na+ absorption. The NHE3 expression section describes apical Na+/H+ exchange, but the passages found do not specify that it exchanges exactly one luminal Na+ for one intracellular H+. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Human intestinal organoid-derived monolayers have been successfully used to study host-pathogen interactions, epithelial barrier function, and various transport processes including secretion of hormones, growth factors, and ions, and absorption of glucose.

    “Human intestinal organoid-derived monolayers have been successfully used to study host–pathogen interactions [10,11,12], epithelial barrier function [13], and various transport processes, including secretion of hormones, growth factors, and ions [14,15,16], and absorption of glucose [17].”
    Quote in the paperSubstantial claim6 of 7 sources checked
    • cites [10] VanDussen, 2015, Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays, Gut source
    • cites [11] Ettayebi, 2016, Replication of human noroviruses in stem cell–derived human enteroids, Science source
    • cites [12] Noel, 2017, A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions, Sci. Rep. source
    • cites [13] Cheng, 2021, Targeting uPA-uPAR interaction to improve intestinal epithelial barrier integrity in inflammatory bowel disease, EBioMedicine source
    • cites [14] Kozuka, 2017, Development and Characterization of a Human and Mouse Intestinal Epithelial Cell Monolayer Platform, Stem Cell Rep. source
    • cites [15] Tse, 2019, cAMP Stimulates SLC26A3 Activity in Human Colon by a CFTR-Dependent Mechanism That Does Not Require CFTR Activity, Cell. Mol. Gastroenterol. Hepatol. source
    • cites [16] Yin, 2018, Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers, Cell. Mol. Gastroenterol. Hepatol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    SGLT1 is a secondary active transporter that translocates two Na ions coupled with one glucose molecule into cells and is the most abundant glucose transporter in the small intestinal brush border membrane.

    “SGLT1 is a secondary active transporter that translocates two Na ions coupled with one glucose molecule into cells and is also the most abundant glucose transporter in the small intestinal BBM [21].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [21] Koepsell, 2020, Glucose transporters in the small intestine in health and disease, Pflügers Arch. Eur. J. Physiol. source

      The SGLT1 section states that SGLT1 is a secondary active transporter that carries one glucose molecule with two sodium ions into cells, and it locates SGLT1 in the brush border membrane (BBM). It reports Sglt1 as the most abundant plasma-membrane protein in mouse small intestine, but does not establish that SGLT1 is the most abundant glucose transporter specifically in the BBM across species. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    siRNA-mediated knockdown is simple, convenient, economical, and effective in all mammalian somatic cell types.

    “Some of the advantages of siRNA-mediated knockdown are that it is simple, convenient, economical, and effective in all mammalian somatic cell types [45].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [45] Teriyapirom, 2021, Genetic engineering in organoids, J. Mol. Med. source

      The Tools for genetic engineering section (page unavailable) says RNAi, including siRNA-mediated knockdown, is effective in all mammalian somatic cells and requires no prior genetic manipulation. It does not describe siRNA knockdown as simple, convenient, or economical; the Discussion instead notes difficulties with siRNA libraries in screens. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Reciprocal regulation between NHE3 and SGLT1 was previously demonstrated in a rat intestinal epithelial cell line (IEC-18).

    “Reciprocal regulation between NHE3 and SGLT1 was previously demonstrated in a rat intestinal epithelial cell line (IEC-18) [27].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [27] Coon, 2011, Reciprocal regulation of the primary sodium absorptive pathways in rat intestinal epithelial cells, Am. J. Physiol. Physiol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Diabetes is secondary to altered glucose homeostasis.

    “Likewise, diabetes is secondary to altered glucose homeostasis [67], and this study suggests a critical role for NHE3 in the regulation of SGLT1, which is the predominant intestinal glucose transporter.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [67] Galicia-Garcia, U., Benito-Vicente, A., Jebari, S., Larrea-Sebal, A., Siddiqi, H., Uribe, K.B., Ostolaza, H., and Martín, C. (2020). Pathophysiology of Type 2 Diabetes Mellitus. Int. J. Mol. Sci., 21. source

  • May combine two claims.

    Premisecitation

    Intracellular cNO differentially regulates NHE3 and SGLT1 in vitro in IEC-18 cells and in vivo in rabbit small intestine.

    “Furthermore, intracellular cNO was shown to differentially regulate NHE3 and SGLT1 in vitro in IEC-18 cells [23,24,25] and in vivo in rabbit small intestine [25,55,56], which implies functional compensation between the two primary Na absorptive pathways.”
    Quote in the paperNot a substantial claim2 of 5 sources checked
    • cites [23] Coon, 2008, Constitutive nitric oxide differentially regulates Na-H and Na-glucose cotransport in intestinal epithelial cells, Am. J. Physiol. Liver Physiol. source
    • cites [24] Palaniappan, 2018, Direct and specific inhibition of constitutive nitric oxide synthase uniquely regulates brush border membrane Na-absorptive pathways in intestinal epithelial cells, Nitric Oxide source

      The Abstract and Discussion report that reducing intracellular cNO in IEC-18 cells inhibits SGLT-1 and stimulates NHE3. The Introduction reports the same opposing effects after pharmacological NO inhibition in rabbits, but cautions that the in vivo observations may reflect effects in multiple tissues; it does not establish intracellular cNO regulation in rabbit epithelial cells. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [25] Palaniappan, 2019, Stimulation of constitutive nitric oxide uniquely and compensatorily regulates intestinal epithelial cell brush border membrane Na absorption, Physiol. Rep. source
    • cites [55] Coon, 2005, Na-glucose and Na-neutral amino acid cotransport are uniquely regulated by constitutive nitric oxide in rabbit small intestinal villus cells, Am. J. Physiol. Liver Physiol. source
    • cites [56] Coon, 2007, Mechanism of regulation of rabbit intestinal villus cell brush border membrane Na/H exchange by nitric oxide, Am. J. Physiol. Liver Physiol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    CRISPR technology has drawbacks including Cas9 off-target activity, cost, and a labor-intensive process of guide RNA design, viral packaging and infection, isolation of single cells, expansion of clones, and verification of correct targeting.

    “While complete gene knockout is often preferred, there are some drawbacks of CRISPR technology including Cas9 off-target activity [46], as well as the cost and labor-intensive process of guide RNA design, viral packaging and infection, isolation of single cells, expansion of clones, and verification of correct targeting [47].”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [46] Sander, 2014, CRISPR-Cas systems for editing, regulating and targeting genomes, Nat. Biotechnol. source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [47] Giuliano, 2019, Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9, Curr. Protoc. Mol. Biol. source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Several Na-dependent nutrient co-transporters including SGLT1 depend on the pump activity of the Na-K-ATPase at the basolateral membrane to maintain low levels of intracellular Na and provide a favorable electrochemical gradient necessary for their functioning.

    “In addition, several Na-dependent nutrient co-transporters including SGLT1 depend on the pump activity of the Na-K-ATPase at the basolateral membrane to maintain low levels of intracellular Na in order to provide a favorable electrochemical gradient necessary for their functioning [53,54].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [53] Thwaites, 2007, H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine, Exp. Physiol. source
    • cites [54] Wright, 2007, Active sugar transport in health and disease, J. Intern. Med. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    A likely reason for the limited use of siRNA technology in 3D organoid cultures is the technical challenge of delivering siRNAs through the extracellular matrix to organoids in situ.

    “The use of siRNA technology in 3D organoid cultures has been very limited [48,49,50], likely also due to the technical challenge of delivering siRNAs through the extracellular matrix to organoids in situ [49].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [49] Morgan, 2018, Optimized delivery of siRNA into 3D tumor spheroid cultures in situ, Sci. Rep. source

  • Classified as probably not a substantial claim.

    Premisecitation

    Organoid-derived 2D monolayer culture models have been instrumental for studies in which access to the apical surface is important.

    “Furthermore, the recent development of organoid-derived 2D monolayer culture models has been instrumental for studies in which access to the apical surface is important [10,11,44].”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [10] VanDussen, 2015, Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays, Gut source
    • cites [11] Ettayebi, 2016, Replication of human noroviruses in stem cell–derived human enteroids, Science source
    • cites [44] In, 2015, Enterohemorrhagic Escherichia coli Reduces Mucus and Intermicrovillar Bridges in Human Stem Cell-Derived Colonoids, Cell. Mol. Gastroenterol. Hepatol. source

  • May combine two claims.

    Premisecitation

    Our laboratory has published evidence that compensatory regulation between the two primary Na absorptive pathways may be lost in obesity, leading to enhanced absorption of both Na and glucose and contributing to hypertension and diabetes as complications of obesity.

    “As previously noted, our laboratory has published evidence that the compensatory regulation between the two primary Na absorptive pathways may be lost in obesity, leading to enhanced absorption of both Na and glucose, thus contributing to the pathophysiology of two common complications of obesity: hypertension and diabetes [8].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [8] Palaniappan, 2019, Inhibition of intestinal villus cell Na/K-ATPase mediates altered glucose and NaCl absorption in obesity-associated diabetes and hypertension, FASEB J. source

  • Classified as probably not a substantial claim.

    Premisereasoning

    Whether NHE3 and SGLT1 regulate one another in normal human enterocytes is unknown, mainly due to a lack of appropriate experimental models.

    “However, whether NHE3 and SGLT1 regulate one another in normal human enterocytes is unknown, mainly due to a lack of appropriate experimental models.”
    • Also stated in: § 1. Introduction · plain
      “However, it is not known if NHE3 and SGLT1 regulate one another in human enterocytes, mainly due to a lack of appropriate experimental models.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Human jejunal organoid-derived monolayers are composed of differentiated enterocytes and are therefore a suitable in vitro model to investigate the regulation of apical Na transporter function in normal human enterocytes.

    “Thus, human jejunal organoid-derived monolayers are composed of differentiated enterocytes and are therefore a suitable in vitro model to investigate the regulation of apical Na transporter function in normal human enterocytes.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Findings in human jejunal organoid-derived monolayers reflect normal human enterocyte physiology in vivo.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Coupled NaCl absorption is achieved by the dual operation of Na-H and Cl-HCO3 exchangers in the brush border membrane of villus cells, and their regulation is mediated by intracellular pH.

    “For example, coupled NaCl absorption is achieved by the dual operation of Na-H and Cl-HCO3 exchangers in the BBM of villus cells, and their regulation is mediated by intracellular pH [51,52].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [51] Zachos, 2005, Molecular Physiology of Intestinal N+/H+ Exchange, Annu. Rev. Physiol. source
    • cites [52] Sundaram, 1991, pH regulation in ileum: Na(+)-H+ and Cl(-)-HCO3- exchange in isolated crypt and villus cells, Am. J. Physiol. Liver Physiol. source

  • May combine two claims.

    Interpretationreasoning

    Reciprocal compensatory regulation between transporters is unique and likely functions in the maintenance of intestinal Na homeostasis under physiological conditions.

    “Reciprocal compensatory regulation between transporters is unique, and likely functions in the maintenance of intestinal Na homeostasis in physiological conditions.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Hypertension results from altered Na homeostasis.

    “For example, hypertension results from altered Na homeostasis [66], which this study suggests is a function of both coupled NaCl absorption (via the dual operation of Na-H and Cl-HCO3 exchangers), and SGLT1.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [66] Jaitovich, 2010, Salt, Na+,K+-ATPase and hypertension, Life Sci. source

  • May combine two claims.

    Premisecitation

    cNO, acting through cGMP/PKG signaling, inhibits NHE3 but stimulates SGLT1 in human jejunal organoid monolayers across multiple human specimens.

    “Consistent with our previous studies, we recently demonstrated that cNO, which mediates its effects through cGMP/PKG signaling, also inhibits NHE3 but stimulates SGLT1 in human jejunal organoid monolayers, across multiple human specimens [18].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [18] Haynes, 2022, Regulation of nutrient and electrolyte absorption in human organoid-derived intestinal epithelial cell monolayers, Transl. Res. source
21 of 21

  • The paper relies on this without support.

    siRNA-mediated knockdown of NHE3 or SGLT1 is specific to the targeted gene and does not produce off-target effects that account for the observed changes in the other transporter.

    “Silencer Select pre-designed siRNA (Ambion, Waltham, MA, USA) targeting human NHE3 (SLC9A3; s13029), human SGLT1 (SLC5A1; s12953), or the non-targeting negative control siRNA (Negative Control No. 1) was used for transfection experiments.”

  • The paper relies on this without support.

    EIPA-sensitive 22Na uptake specifically measures NHE3 activity in human jejunal organoid-derived monolayers.

    “NHE3 activity was measured directly as the Na-dependent, 5-(N-Ethyl-N-isopropyl)amiloride (EIPA)-sensitive uptake of the radioisotope 22Na.”

  • The paper relies on this without support.

    Phlorizin-sensitive 3H-OMG uptake specifically measures SGLT1 activity in human jejunal organoid-derived monolayers.

    “SGLT1 activity was measured directly as the Na-dependent, phlorizin-sensitive uptake of the radiolabeled glucose analogue 3H-3-O-Methyl-D-glucopyranose (3H-OMG).”

  • The paper relies on this without support.

    Housekeeping genes ACTB and TBP are stably expressed across the experimental conditions and are valid for normalization.

    “Housekeeping genes ACTB and TBP were used for normalization.”

  • The paper relies on this without support.

    The two-tailed Student's t test assumptions of normal distribution and equal variance are met by the data.

    “p values were derived via a two-tailed Student’s t test using Prism 9 software (GraphPad, San Diego, CA, USA). Differences were considered statistically significant when p < 0.05.”

  • The paper relies on this without support.

    Findings in human jejunal organoid-derived monolayers reflect normal human enterocyte physiology in vivo.

    “Thus, human jejunal organoid-derived monolayers are composed of differentiated enterocytes and are therefore a suitable in vitro model to investigate the regulation of apical Na transporter function in normal human enterocytes.”

  • The paper relies on this without support.

    siRNA technology had not been previously demonstrated in organoid-derived monolayer cultures.

    “While siRNA technology is routinely used to induce transient gene silencing in immortalized cell lines, it has not been previously demonstrated in organoid-derived monolayer cultures.”

  • The paper relies on this without support.

    The custom anti-SGLT1 antibody specifically detects SGLT1 in Western blots.

    “Polyclonal antibodies against SGLT1 were raised in rabbit using Invitrogen (Waltham, MA, USA) custom antibody services. A peptide corresponding to amino acids 581–599 of human SGLT1 (QEGPKETIEIETQVPEKKK) was used as the immunogen [28] and for antibody affinity purification.”

  • The paper relies on this without support.

    siRNA-mediated gene knockdown in organoid-derived monolayers has not been reported.

    “Moreover, siRNA-mediated gene knockdown in organoid-derived monolayers has not been reported.”

  • The paper relies on this without support.

    siRNA is often difficult in nonimmortalized cell lines and primary cells due to low transfection efficiency.

    “While the use of siRNA for gene knockdown is common in 2D immortalized cell lines, it is often difficult in nonimmortalized cell lines and primary cells due to low transfection efficiency.”

  • The paper relies on this without support.

    SGLT1 is the predominant intestinal glucose transporter.

    “which is the predominant intestinal glucose transporter.”
11 of 11

Cites

Flagged first, then by citations

2 of the 68 references couldn’t be matched to a known paper.

Research works printed but not linked: 2

  1. Said, H.M. (2018). Chapter 57—Intestinal Anion Absorption. Physiology of the Gastrointestinal Tract, Academic Press. [6th ed.].
  2. Said, H.M. (2018). Chapter 56—Na+/H+ Exchange in Mammalian Digestive Tract. Physiology of the Gastrointestinal Tract, Academic Press. [6th ed.].

Cited by

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Related

Authors
Haynes, J., Palaniappan, B., Crutchley, J. M., & Sundaram, U.
Affiliations
Marshall University (US)
Corresponding author
Sundaram, Uma
ORCID
Venue
Cells
Publisher
MDPI AG
Published
2024-09-28
Volume
13
Issue
19
Pages
1623
ISSN
2073-4409 (ISSN-L)
DOI
10.3390/cells13191623
PMC
PMC11482628
OpenAlex
W4402997507
Semantic Scholar
22ce9e51d6ce3f4a3c30f68ba7cafcb66cf4f412
Cited by
2
References
68
Citations by year
2026: 1 · 2024: 1
FWCI
0.23
Type
journal article
Language
en
Open access
Licence
Locations
  • Cells (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Institutes of Health grants (DK-67420, DK-108054, P20GM121299-01A1, BX003443-01)
  • Veteran’s Administration Merit Review grant (DK-67420, DK-108054, P20GM121299-01A1, BX003443-01)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion Transport and Channel Regulation
  • Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Digestive system and related health
Keywords
sodium uptake, NHE3, SGLT1, compensatory regulation, siRNA knockdown, human small intestine, Na+/glucose cotransport
MeSH terms
Sodium-Hydrogen Exchanger 3, Humans, Jejunum, Microvilli, Organoids, Sodium, Sodium-Hydrogen Exchangers, Enterocytes, RNA, Small Interfering, Sodium-Glucose Transporter 1

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.

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