NHE3 Expression Research Articles

Osmoregulation and reproduction: evolutionary trends in prolactin functions from fish to mammals

The study of prolactin function evolution provides key insights into the diverse effects of this hormone in mammals, both in health and disease, which is relevant from both theoretical and practical perspectives. This article reviews both original and literature data concerning the role of prolactin and its receptors in regulating the sexual dimorphism of freshwater adaptation in the three-spined stickleback Gasterosteus aculeatus L. It is demonstrated that mRNA expression of prolactin gene 1 (one of two prolactin paralogs) and its receptor PRLRA increases in the brains of female sticklebacks only upon transitioning to freshwater. The brain and kidneys of sticklebacks, as androgen-dependent organs, exhibit sex-dependent expression of Prlra in seawater. It is suggested that sex-dependent osmoregulatory effects of prolactin are mediated through the PRLRA receptor in these organs. The PRLRB receptor, expressed in the kidneys and brains of sticklebacks regardless of sex in seawater, shows increased sensitivity to reduced salinity, suggesting a more active role in implementing sex-independent osmoregulatory functions of prolactin. Gills and intestines, as osmoregulatory organs, express the PRLRA and PRLRB receptors independent of sex in both seawater and freshwater. With freshwater adaptation, there is a concurrent increase in the expression of Prl1 in the brains of females and the expression of Atp1a1 (α1a subunit of Na+/K+-ATPase), Nhe3 (NHE3 sodium-proton antiport gene), and Ecac (epithelial calcium channel gene) in their gills. It is presumed that these gill genes are under positive control by prolactin. Exploring the potential for prolactin’s osmoregulatory function in mammals revealed that it may manifest in conditions such as pathologies accompanied by increased expression of prolactin receptor isoforms in osmoregulatory organs. One of such pathologies is cholestasis in female rats, which was associated with an increase in Prlr isoform expression and changes in activity and ratio of Na+/K+-ATPase subunits in the kidney. Thus, it is concluded that in fish, the osmoregulatory function of prolactin is sex-dependent, while in mammals, it may manifest under conditions of disrupted water-salt exchange.

Effects of Dapagliflozin in Combination with Hydrochlorothiazide on Cardiac Remodeling in Rats with Heart Failure After Myocardial Infarction.

In addition to its antihypertensive and diuretic effects, hydrochlorothiazide also demonstrates additional cardioprotective properties; however, the existence of a synergistic interaction between dapagliflozin and hydrochlorothiazide remains unclear.To establish a rat model of heart failure for investigating the effects and mechanisms of dapagliflozin in combination with hydrochlorothiazide during early intervention, H9c2 cells were cultured to validate their in vitro efficacy. The combination group exhibits a synergistic improvement in hemodynamics, ejection fraction, and a reduction in plasma B-type natriuretic peptide concentration. This combination effectively decreases collagen volume fraction and the expression of collagen I and III, p47phox, p67phox, NF-κB p65, Bax, and caspase-3. The combination group demonstrates a synergistic effect in enhancing cardiac function, attenuating oxidative stress and inflammation. The in vitro effects of the combination were demonstrated in H9c2 cardiomyocytes. In addition, the combination exhibits a more pronounced inhibitory effect on NHE1 expression. The expression of NHE1 in H9c2 cells is inhibited by hydrochlorothiazide, thereby alleviating the consequences of NHE1 overexpression. The results of molecular docking and kinetic simulations indicate a strong binding affinity (-6.1 kcal/mol) between hydrochlorothiazide and NHE1, resulting in the formation of a stable conformation. This may elucidate the underlying mechanism responsible for the synergistic effects of drugs.The combination of dapagliflozin and hydrochlorothiazide has synergistic effects on improving cardiac function, oxidative stress, and inflammation in rats with heart failure. Hydrochlorothiazide binds to and inhibits the expression of NHE1, thereby enhancing dapagliflozin's inhibitory effect on NHE activity. This mechanism potentially elucidates its enhanced cardioprotective effects.

Abstract MP33: A20 in the Kidney Epithelium Attenuates Angiotensin II-induced Hypertension by Constraining Renal Tubular NHE3 Expression

Whereas the ubiquitin editor A20 limits NF-κB-mediated signaling in myeloid leukocytes, the functions of A20 in renal epithelial cells during hypertension have not been described. We recently found that A20 in the kidney tubule ameliorates hypertension and cardiac hypertrophy induced by chronic angiotensin (Ang) II infusion (500ng/kg/min) in mice. Here we report that following 3 weeks of hypertension, the kidneys of mice with tubular deletion of A20 in the kidney epithelium (“A20 iKKO” = A20 flox/flox Pax8-rtTA TetO-Cre + ) have augmented protein expression of the proximal tubular sodium transporter NHE3 (1.5±0.1 vs. 1.0±0.1; p <0.01) but similar levels of ENaC subunit expression in the collecting tubule ( p =NS for all 3 subunits) vs wild-type (WT) controls. Moreover, kidneys from A20 iKKO mice have upregulated mRNA levels for the T cell chemokine CCL5 (2.4±0.3 vs. 1.0±0.3; p <0.01) and higher proportions of effector memory CD8 + T cells (51.2±2.3 vs. 39.3±2.9 % of CD8 + ; p =0.013) compared to WTs despite similar numbers of effector memory T cells in the spleen ( p =NS). To examine whether A20 in the kidney attenuates hypertension by constraining tubular NHE3 expression, we bred A20 iKKO mice with an NHE3 flox/flox line, thereby generating mice with double A20 and NHE3 deletion in the kidney tubule (“Dual KKO”) mice. At baseline, kidneys from Dual KKO mice showed robust reductions in mRNA for A20 (0.5±0.1 vs. 1.0±0.1; p =0.022) and NHE3 (0.2±0.05 vs. 1.0±0.15; p =0.002) compared to WT littermates. During the first ten days of Ang II infusion, mean arterial pressures (MAPs) in the Dual KKO mice were lower than the MAPs previously recorded in our A20 iKKO cohort (129±3 vs. 152±4 mmHg; p <0.01). Moreover, during 3 weeks of Ang II infusion, Dual KKO and WT mice maintained similar MAPs (145±7 vs. 149±13 mmHg; p =0.78), and heart weight to body weight ratios (5.9±0.3 vs. 5.5±0.2 mg/g; p =0.37) in concomitant measurements. Thus, A20 in the kidney tubule blunts the chronic hypertensive response by curtailing NHE3 expression. Our data further suggest that A20 in the nephron can blunt renal inflammation without altering systemic T cell immunity. Stimulating the A20 signaling pathway in the kidney may thus attenuate NHE3-dependent hypertension without conferring attendant risks of global immunosuppression.

NHE1 regulation in NAFLD in vitro contributes to hepatocyte injury and HSC crosstalk.

Non-alcoholic fatty liver disease (NAFLD) is the fastest growing cause of liver-associated death globally. Whole-body knockout (KO) of Na+/H+ exchanger 1 (NHE1, SLC9A1) was previously proposed to protect against high fat diet-induced liver damage, however, mechanistic insight was lacking. The aim of the present work was to address this question in vitro to determine how NHE1 specifically in hepatocytes impacts lipid overload-induced inflammation, fibrosis, and hepatocyte- hepatic stellate cell (HSC) crosstalk. We induced palmitate (PA)-based steatosis in AML12 and HepG2 hepatocytes, manipulated NHE1 activity pharmacologically and by CRISPR-Cas knockout (KO) and -overexpression, and measured intracellular pH (pHi), steatosis-associated inflammatory and fibrotic mediators and cell death. PA treatment increased NHE1 mRNA levels but modestly reduced NHE1 protein expression and hepatocyte pHi. NHE1 KO in hepatocytes did not alter lipid droplet accumulation but reduced inflammatory signaling (p38 MAPK activity), lipotoxicity (4-HNE accumulation) and apoptosis (PARP cleavage). Conditioned medium from PA-treated hepatocytes increased expression of NHE1 and of the fibrosis regulator tissue inhibitor of matrix metalloproteases-2 (TIMP2) in LX-2 HSCs, in a manner abolished by NHE1 KO in hepatocytes. We conclude that NHE1 is regulated in NAFLD in vitro and contributes to the ensuing damage by aggravating hepatocyte injury and stimulating hepatocyte-HSC crosstalk.

Na+/H+-exchange inhibition by cariporide is compensated via Na+,HCO3−-cotransport and has no net growth consequences for ErbB2-driven breast carcinomas

Defense against intracellular acidification of breast cancer tissue depends on net acid extrusion via Na+,HCO3−-cotransporter NBCn1/Slc4a7 and Na+/H+-exchanger NHE1/Slc9a1. NBCn1 is increasingly recognized as breast cancer susceptibility protein and promising therapeutic target, whereas evidence for targeting NHE1 is discordant. Currently, selective small molecule inhibitors exist against NHE1 but not NBCn1. Cellular assays—with some discrepancies—link NHE1 activity to proliferation, migration, and invasion; and disrupted NHE1 expression can reduce triple-negative breast cancer growth. Studies on human breast cancer tissue associate high NHE1 expression with reduced metastasis and—in some molecular subtypes—improved patient survival. Here, we evaluate Na+/H+-exchange and therapeutic potential of the NHE1 inhibitor cariporide/HOE-642 in murine ErbB2-driven breast cancer. Ex vivo, cariporide inhibits net acid extrusion in breast cancer tissue (IC50 = 0.18 μM) and causes small decreases in steady-state intracellular pH (pHi). In vivo, we deliver cariporide orally, by osmotic minipumps, and by intra- and peritumoral injections to address the low oral bioavailability and fast metabolism. Prolonged cariporide administration in vivo upregulates NBCn1 expression, shifts pHi regulation towards CO2/HCO3−-dependent mechanisms, and shows no net effect on the growth rate of ErbB2-driven primary breast carcinomas. Cariporide also does not influence proliferation markers in breast cancer tissue. Oral, but not parenteral, cariporide elevates serum glucose by ∼1.5 mM. In conclusion, acute administration of cariporide ex vivo powerfully inhibits net acid extrusion from breast cancer tissue but lowers steady-state pHi minimally. Prolonged cariporide administration in vivo is compensated via NBCn1 and we observe no discernible effect on growth of ErbB2-driven breast carcinomas.

Lipopolysaccharide Triggers Luminal Acidification to Promote Defense Against Bacterial Infection in Vaginal Epithelium

The vaginal epithelium plays pivotal roles in host defense against pathogen invasion, contributing to the maintenance of an acidic microenvironment within the vaginal lumen through the activity of acid-base transport proteins. However, the precise defense mechanisms employed by the vaginal epithelium following bacterial infection remain incompletely understood. This study demonstrated that the bacterial lipopolysaccharide (LPS) potentiated net proton efflux by up-regulating the expression of Na+-H+ exchanger 1 (NHE1), without affecting other acid-base transport proteins in vaginal epithelial cells. Pharmacological inhibition or genetic knockdown of Toll-like receptor-4 (TLR4) and the extracellular-signal-regulated protein kinase (ERK) signaling pathway effectively counteracted the up-regulation of NHE1 and the enhanced proton efflux triggered by LPS in vaginal epithelial cells. In vivo studies revealed that LPS administration led to luminal acidification through the up-regulation of NHE1 expression in the rat vagina. Moreover, inhibition of NHE exhibited impaired defense against acute bacterial infection in the rat vagina. These findings collectively indicated the active involvement of vaginal epithelial cells in facilitating luminal acidification during acute bacterial infection, offering potential insights into the treatment of bacterial vaginosis.

SGLT2i Alleviates Atherosclerosis by Inhibiting NHE1 Activation to Protect against Macrophage Senescence Induced by Angiotensin II.

Sodium-dependent glucose transporter (SGLT2) inhibitors (SGLT2i) have been found to have anti-atherosclerotic effects in clinical treatment. The aim of this study was to explore whether angiotensin II (Ang II) induces changes in the expression of Na+/H+ exchanger of cytoplasmic membrane channel proteins (NHE1) and SGLT2 in macrophages and whether dapagliflozin (DAPA), an SGLT2i, protects against Ang II induced macrophage senescence by inhibiting NHE1 activation to alleviate Atherosclerosis (AS). After intervention with DAPA plus gavage or feeding them a high-fat diet, the mice's aortas were dissected, and oil red O staining was performed. Cell proliferation and toxicity detection, western blot, immunofluorescence, and β-galactosidase staining methods were adopted to detect cell activity, expressions of senescence-related genes, and number of senescent cells after different concentrations of Ang II or DAPA or plasmid NHE1 were treated with RAW264.7 cells. (1) The formation of AS plaques in ApoE -/- mice showed a downward trend under DAPA. (2) After the intervention of Ang II, the cell activity of RAW264.7 decreased, and the expression of senescent cells and related genes increased. (3) Under the Ang II condition, the expression of SGLT2 and NHE1 increased, and SGLT2, NHE1, and senescence-related genes decreased with the addition of DAPA. (4) The expression of NHE1, senescent cells and related genes decreased in RAW264.7 cells after DAPA treatment with plasmid NHE1 intervention. SGLT2i alleviates atherosclerosis by inhibiting NHE1 activation to protect against macrophage senescence induced by Ang II.

Type 1 diabetes human enteroid studies reveal major changes in the intestinal epithelial compartment

Lack of understanding of the pathophysiology of gastrointestinal (GI) complications in type 1 diabetes (T1D), including altered intestinal transcriptomes and protein expression represents a major gap in the management of these patients. Human enteroids have emerged as a physiologically relevant model of the intestinal epithelium but establishing enteroids from individuals with long-standing T1D has proven difficult. We successfully established duodenal enteroids using endoscopic biopsies from pediatric T1D patients and compared them with aged-matched enteroids from healthy subjects (HS) using bulk RNA sequencing (RNA-seq), and functional analyses of ion transport processes. RNA-seq analysis showed significant differences in genes and pathways associated with cell differentiation and proliferation, cell fate commitment, and brush border membrane. Further validation of these results showed higher expression of enteroendocrine cells, and the proliferating cell marker Ki-67, significantly lower expression of NHE3, lower epithelial barrier integrity, and higher fluid secretion in response to cAMP and elevated calcium in T1D enteroids. Enteroids established from pediatric T1D duodenum identify characteristics of an abnormal intestinal epithelium and are distinct from HS. Our data supports the use of pediatric enteroids as an ex-vivo model to advance studies of GI complications and drug discovery in T1D patients.

Sex-dependent polymorphism in urinary output is reversed upon deletion of exchange protein directly activated by cAMP (Epac)

It is well-described that there are notable sex-dependent differences in urinary production with females having a smaller volume and greater urinary concentrating ability. Apart from contribution of the androgenic inputs, the underlying signaling intermediates remain largely unexplored. We previously showed that exchange protein directly activated by cAMP (Epac) is a critical regulator of water-solute transport in the proximal tubule and collecting duct. Deletion of either Epac1 or Epac2 isoforms increases urinary output and Na+ excretion. Here, we used metabolic cage balance studies to test whether Epac signaling mediates sex-dependent differences in urinary production. We found that concomitant deletion of Epac1&2 moderately increases 24h urinary volume by 38% (from 1.82+/-0.13 to 2.52+/-0.21 ml) in males, but drastically augment urinary production in females by 320% (from 1.39+/-0.10 to 4.45+/-0.37 ml). This was associated with only a mild though significant reduction in urinary osmolarity in males by 35%, whereas urine osmolarity was markedly decreased from 2511+/-5 to 977+/-60 mOsm in females. Consistently, plasma osmolarity was not different in EpacWT versus Epac1&2−/− males, but became significantly more hypertonic in Epac1&2−/− females. We further determine the functional consequences of impaired Epac cascade on fluid reabsorption in the proximal tubule and collecting duct in males and females. Deletion of Epac1 and Epac2 decreases NHE-3 expression in the proximal tubule in an additive manner. In addition, Na+/H+-dependent recovery after acidification was drastically reduced in freshly isolated split-opened proximal tubules from Epac1&2−/− mice when compared to EpacWT. Interestingly, the reduction of NHE-3 expression was more pronounced in Epac1&2−/− females (by 89%) than in males (by 59%). Immunofluorescence confocal images revealed much more diffuse intracellular distribution of AQP2 water channel in Epac1&2−/− females when compared to males. Furthermore, osmotically-driven water influx was reduced in principal cells within split-opened collecting ducts of Epac1&2−/− females but not males, when compared to respective EpacWT. In summary, our results reveal a substantially greater role of Epac signaling in renal water handling in females than in males. Interestingly, Epac1&2 deletion leads to a reversed pattern of sexual dimorphism in urinary production with females excreting larger volumes of hypotonic urine than males. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex-Based Differences in Proximal Tubule Endocytic Capacity in Mice

Women are more protected from acute kidney injury, and chronic kidney disease (CKD) in women progresses more slowly to end-stage renal disease compared to men. CKD involves changes in the integrity of the glomerular barrier function with consequent proteinuria. The kidney proximal tubule (PT) normally reclaims albumin and other filtered proteins via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. PT health, and ultimately kidney function, is impacted by the uptake of excess albumin and by nephrotoxic drugs that enter cells in a megalin/cubilin dependent manner. Recent transcriptomic studies in mice have shown differences in the expression of megalin and cubilin between males and females and across the S1, S2, and S3 sub-segments that comprise the PT. Additionally, reduced reabsorption of water and salt by the PT has been demonstrated in female rodents. These differences in PT receptor expression and functionality likely contribute to sex-based differences in PT endocytic capacity. To determine if there are sex-based differences in the recovery of filtered proteins by the PT, urine and kidney tissue were collected from five male and five female 13-week-old C57BL/6 mice. Urinary albumin was quantified by ELISA. Kidneys were processed for Western blotting and immunofluorescence staining. We used imaging-based approaches to quantify the expression and distribution of albumin, megalin, and cubilin in SGLT2-positive S1 and SGLT2-negative, OAT1-positive S2 segments. Under normal conditions, S1 had greater intensity of subapical albumin staining in both males and females. However, females had greater intensity of albumin staining in S2 normalized to S1 compared to males. Female mice had lower levels of urinary albumin excretion when normalized to creatinine than males. Female mice had greater expression of total megalin (1.5x) and cubilin (1.4x) and decreased expression of NHE3 (0.7x) in the kidney cortex. Male and female mice had similar levels of megalin expression and surface localization in S1, however female mice had greater expression and surface localization of megalin in S2. Males had greater colocalization of megalin with lysosomal marker, LAMP1. In both males and females, the colocalization of megalin with LAMP1 decreased from S1 to S2. Together, these data suggest that, at baseline in female mice, S2 has greater endocytic capacity and contributes more to the uptake of normally filtered albumin than in males. This increased capacity could explain the reduced urinary excretion of albumin observed in rodent injury models and in women with CKD. Current studies are focused on incorporating these sex-based differences into a multiscale mathematical model of protein uptake along the length of the PT. National Institutes of Health: T32 DK007052, F31 DK121394, R01 DK125049, R01 DK118726, R01 DK083785, S10 OD021627, S10 OD028596, U54 DK137329, ASN Foundation for Kidney Research Pre-Doctoral Fellowship Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CGMP-dependent kinase 2, Na+/H+ exchanger NHE3, and PDZ-adaptor NHERF2 co-assemble in apical membrane microdomains.

Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.

New functions and roles of the Na+-H+-exchanger NHE3.

The sodium/proton exchanger isoform 3 (NHE3) is expressed in the intestine and the kidney, where it contributes to hydrogen secretion and sodium (re)absorption. The roles of this transporter have been studied by the use of the respective knockout mice and by using pharmacological inhibitors. Whole-body NHE3 knockout mice suffer from a high mortality rate (with only ∼30% of mice surviving into adulthood), and based on the expression of NHE3 in both intestine and kidney, some conclusions that were originally derived were based on this rather complex phenotype. In the last decade, more refined models have been developed that added temporal and spatial control of NHE3 expression. For example, novel mouse models have been developed with a knockout of NHE3 in intestinal epithelial cells, tubule/collecting duct of the kidney, proximal tubule of the kidney, and thick ascending limb of the kidney. These refined models have significantly contributed to our understanding of the role of NHE3 in a tissue/cell type-specific manner. In addition, tenapanor was developed, which is a non-absorbable, intestine-specific NHE3 inhibitor. In rat and human studies, tenapanor lowered intestinal Pi uptake and was effective in lowering plasma Pi levels in patients on hemodialysis. Of note, diarrhea is seen as a side effect of tenapanor (with its indication for the treatment of constipation) and in intestine-specific NHE3 knockout mice; however, effects on plasma Pi were not supported by this mouse model which showed enhanced and not reduced intestinal Pi uptake. Further studies indicated that the gut microbiome in mice lacking intestinal NHE3 resembles an intestinal environment favoring the competitive advantage of inflammophilic over anti-inflammatory species, something similar seen in patients with inflammatory bowel disease. This review will highlight recent developments and summarize newly gained insight from these refined models.

Myometrium infection decreases TREK1 through NHE1 and increases contraction in pregnant mice.

Intrauterine infection during pregnancy can enhance uterine contractions. A two-pore K+ channel TREK1 is crucial for maintaining uterine quiescence and reducing contractility, with its properties regulated by pH changes in cell microenvironment. Meanwhile, the sodium hydrogen exchanger 1 (NHE1) plays a pivotal role in modulating cellular pH homeostasis, and its activation increases smooth muscle tension. By establishing an infected mouse model of Escherichia coli (E. coli) and lipopolysaccharide (LPS), we used Western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence to detect changes of TREK1 and NHE1 expression in the myometrium, and isometric recording measured the uterus contraction. The NHE1 inhibitor cariporide was used to explore the effect of NHE1 on TREK1. Finally, cell contraction assay and siRNA transfection were performed to clarify the relationship between NHE1 and TREK1 in vitro. We found that the uterine contraction was notably enhanced in infected mice with E. coli and LPS administration. Meanwhile, TREK1 expression was reduced, whereas NHE1 expression was upregulated in infected mice. Cariporide alleviated the increased uterine contraction and promoted myometrium TREK1 expression in LPS-injected mice. Furthermore, suppression of NHE1 with siRNA transfection inhibited the contractility of uterine smooth muscle cells and activated the TREK1. Altogether, our findings indicate that infection increases the uterine contraction by downregulating myometrium TREK1 in mice, and the inhibition of TREK1 is attributed to the activation of NHE1.NEW & NOTEWORTHY Present work found that infection during pregnancy will increase myometrium contraction. Infection downregulated NHE1 and followed TREK1 expression and activation decrease in myometrium, resulting in increased myometrium contraction.

Alleviating effect of Lactobacillus pentosus CQZC01 probiotic strain on antibiotic-induced diarrhea in mice

This experiment used mice as the experimental subjects to study the alleviating effect of Lactobacillus pentosus CQZC01 (LPCQZC01) on antibiotic-induced diarrhea in mice. After adaptive feeding for seven days, Kunming (KM) mice were randomly divided into the following groups: positive control group, model group, normal group, high concentration group of LPCQZC01 (H-LPCQZC01, 1×109 CFU/mL), and low concentration group of LPCQZC01 (L-LPCQZC01, 1×108 CFU/mL). Except for the normal group, mice were orally administered with lincomycin hydrochloride (120 mg/day) for 7 consecutive days. Changes in body weight, food intake, water consumption, and fecal water content of mice were observed. A histological examination of mouse colon and small intestine sections was done to find out if Lactobacillus pentosus CQZC01 may help mice with antibiotic-induced diarrhea. The findings demonstrated that, in comparison to the model group, mice in the H-LPCQZC01 and L-LPCQZC01 groups had considerably lower blood levels of nterleukin-6 (IL-6), interleukin-17A (IL-17A), 5-hydroxytryptamine (5-HT), and malondialdehyde (MDA), and other variables. In comparison to the model group, the fecal water content in the H-LPCQZC01 and L-LPCQZC01 groups was considerably lower. The H-LPCQZC01 and L-LPCQZC01 groups consumed more food and liquids than the model group did. The H-LPCQZC01 and L-LPCQZC01 groups had more intact colon walls and more compact, regular, and organized intestinal villi. H-LPCQZC01 and L-LPCQZC01 groups had the lower CFTR, EGFR expression and stronger NHE1, NHE2 expression than model group. This study reveals that Lactobacillus pentosus CQZC01 may treat inflammatory lesions in the mouse intestines and successfully treat antibiotic-induced diarrhea in mice.

Compound Chinese medicine (F1) improves spleen deficiency diarrhea by protecting the intestinal mucosa and regulating the intestinal flora.

Compound Chinese medicine (F1) is a traditional prescription in Chinese medicine that is commonly used to treat spleen deficiency diarrhea (SDD). It has demonstrated remarkable effectiveness in clinical practice. However, the precise mechanism by which it exerts its antidiarrheal effect is still unclear. This study aimed at investigating the antidiarrheal efficacy and mechanism of F1 on senna-induced secretory diarrhea (SDD). Senna was utilized to induce the development of a mouse model of senna-induced secretory diarrhea (SDD) in order to observe the rate of diarrhea, diarrhea index, blood biochemistry, and histopathological changes in the small intestine. Additionally, the levels of sodium and hydrogen exchange protein 3 (NHE3) and short-chain fatty acids (SCFAs) were determined using enzyme-linked immunosorbent assay (ELISA). The impact of F1 on the senna-induced SDD mouse models was evaluated by monitoring changes in the gut microbiota through 16S rRNA (V3-V4) sequencing. The results demonstrated that F1, a traditional Chinese medicine, effectively increased the body weight of SDD mice and reduced the incidence of diarrhea and diarrhea index. Additionally, F1 restored liver and kidney function, reduced the infiltration of inflammatory cells in intestinal tissue, and promoted the growth of intestinal villi. Furthermore, F1 was found to enhance the expression of NHE3 and SCFAs. It also increased the abundance of Firmicutes and Lactobacillus species, while decreasing the abundance of Proteobacteria and Shigella.

Reduced surface pH and upregulated AE2 anion exchange in SLC26A3-deleted polarized intestinal epithelial cells.

Loss of function mutations in the SLC26A3 gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question, SLC26A3 was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pHi and apical and basolateral Cl-/HCO3- exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl-/HCO3- exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pHi was not significantly different from that of WT cells, but basolateral Cl-/HCO3- exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl-/HCO3- exchange rate and apical surface pH, but sustained a normal steady-state pHi due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide.NEW & NOTEWORTHY SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl-/HCO3- exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.

Transcriptional regulation of esophageal, intestinal, and branchial solute transporters by salinity, growth hormone, and cortisol in Atlantic salmon.

In marine habitats, Atlantic salmon (Salmo salar) imbibe seawater (SW) to replace body water that is passively lost to the ambient environment. By desalinating consumed SW, the esophagus enables solute-linked water absorption across the intestinal epithelium. The processes underlying esophageal desalination in salmon and their hormonal regulation during smoltification and following SW exposure are unresolved. To address this, we considered whether two Na+ /H+ exchangers (Nhe2 and -3) expressed in the esophagus contribute to the uptake of Na+ from lumenal SW. There were no seasonal changes in esophageal nhe2 or -3 expression during smoltification; however, nhe3 increased following 48 h of SW exposure in May. Esophageal nhe2, -3, and growth hormone receptor b1 were elevated in smolts acclimated to SW for 2.5 weeks. Treatment with cortisol stimulated branchial Na+ /K+ -ATPase (Nka) activity, and Na+ /K+ /2Cl- cotransporter 1 (nkcc1), cystic fibrosis transmembrane regulator 1 (cftr1), and nka-α1b expression. Esophageal nhe2, but not nhe3 expression, was stimulated by cortisol. In anterior intestine, cortisol stimulated nkcc2, cftr2, and nka-α1b. Our findings indicate that salinity stimulates esophageal nhe2 and -3, and that cortisol coordinates the expression of esophageal, intestinal, and branchial solute transporters to support the SW adaptability of Atlantic salmon.

Compromised NHE8 Expression Is Responsible for Vitamin D-Deficiency Induced Intestinal Barrier Dysfunction.

Objectives: Vitamin D (VitD) and Vitamin D receptor (VDR) are suggested to play protective roles in the intestinal barrier in ulcerative colitis (UC). However, the underlying mechanisms remain elusive. Evidence demonstrates that Na+/H+ exchanger isoform 8 (NHE8, SLC9A8) is essential in maintaining intestinal homeostasis, regarded as a promising target for UC therapy. Thus, this study aims to investigate the effects of VitD/VDR on NHE8 in intestinal protection. Methods: VitD-deficient mice, VDR-/- mice and NHE8-/- mice were employed in this study. Colitis mice were established by supplementing DSS-containing water. Caco-2 cells and 3D-enteroids were used for in vitro studies. VDR siRNA (siVDR), VDR over-expression plasmid (pVDR), TNF-α and NF-κb p65 inhibitor QNZ were used for mechanical studies. The expression of interested proteins was detected by multiple techniques. Results: In colitis mice, paricalcitol upregulated NHE8 expression was accompanied by restoring colonic mucosal injury. In VitD-deficient and VDR-/- colitis mice, NHE8 expression was compromised with more serious mucosal damage. Noteworthily, paricalcitol could not prevent intestinal barrier dysfunction and histological destruction in NHE8-/- mice. In Caco-2 cells and enteroids, siVDR downregulated NHE8 expression, further promoted TNF-α-induced NHE8 downregulation and stimulated TNF-α-induced NF-κb p65 phosphorylation. Conversely, QNZ blocked TNF-α-induced NHE8 downregulation in the absence or presence of siVDR. Conclusions: Our study indicates depressed NHE8 expression is responsible for VitD-deficient-induced colitis aggravation. These findings provide novel insights into the molecular mechanisms of VitD/VDR in intestine protection in UC.

Herbal medicine (Oryeongsan) for fluid and sodium balance in renal cortex of spontaneously hypertensive rats

BackgroundHerbal medicine Oryeongsan (ORS), also known as Wulingsan in Chinese, has been used for the treatment of impaired body fluid balance. However, the mechanisms involved are not clearly defined. The purpose of the present study was to identify the actions of ORS on the renal excretory function and blood pressure (BP) and to define the mechanisms involved in association with renin-angiotensin system (RAS) and natriuretic peptide system (NPS) in spontaneously hypertensive rats (SHR), an animal model of human essential hypertension. MethodsChanges in urine volume (UV), excretion of electrolytes including Na+ (urinary excretion of Na+ (UNaV)) were measured. RT-PCR was performed to trace the changes in expression of RAS, NPS and sodium (Na+)-hydrogen (H+) exchanger 3 (NHE3) in the renal cortex. ResultsIn the SHR treated with vehicle (SHR-V) group, UV and UNaV were suppressed and the Na+ balance was maintained at the higher levels leading to an increase in BP compared to WKY-V group. These were accompanied by an increase in NHE3 expression with an accentuation of angiotensin I converting enzyme-angiotensin II type 1 (ACE-AT1) receptor and concurrent suppression of angiotensin II type 2 (AT2) receptor/ACE2-Mas receptor expression in the renal cortex. Chronic treatment with ORS increased UV and UNaV, and decreased the Na+ and water balance with a decrease in BP in the ORS-treated SHR-ORS group compared to SHR-V. These were accompanied by a decrease in NHE3 expression with a suppression of ACE-AT1 receptor and concurrent accentuation of AT2/ACE2-Mas receptor. ConclusionThe present study shows that ORS reduced BP with a decrease in Na+ and water retention by a suppression of NHE3 expression via modulation of RAS and NPS in SHR. The present study provides pharmacological rationale for the treatment of hypertension with ORS in SHR.

Porcine epidemic diarrhea virus causes diarrhea by activating EGFR to regulates NHE3 activity and mobility on plasma membrane.

As part of the genus Enteropathogenic Coronaviruses, Porcine Epidemic Diarrhea Virus (PEDV) is an important cause of early diarrhea and death in piglets, and one of the most difficult swine diseases to prevent and control in the pig industry. Previously, we found that PEDV can block Na+ absorption and induce diarrhea in piglets by inhibiting the activity of the sodium-hydrogen ion transporter NHE3 in pig intestinal epithelial cells, but the mechanism needs to be further explored. The epidermal growth factor receptor (EGFR) has been proved to be one of the co-receptors involved in many viral infections and a key protein involved in the regulation of NHE3 activity in response to various pathological stimuli. Based on this, our study used porcine intestinal epithelial cells (IPEC-J2) as an infection model to investigate the role of EGFR in regulating NHE3 activity after PEDV infection. The results showed that EGFR mediated viral invasion by interacting with PEDV S1, and activated EGFR regulated the downstream EGFR/ERK signaling pathway, resulting in decreased expression of NHE3 and reduced NHE3 mobility at the plasma membrane, which ultimately led to decreased NHE3 activity. The low level of NHE3 expression in intestinal epithelial cells may be a key factor leading to PEDV-induced diarrhea in newborn piglets. This study reveals the importance of EGFR in the regulation of NHE3 activity by PEDV and provides new targets and clues for the prevention and treatment of PEDV-induced diarrhea in piglets.