NHE3 mRNA Research Articles

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.

Role of KDM2B epigenetic factor in regulating calcium signaling in prostate cancer cells

KDM2B, a histone lysine demethylase, is expressed in a plethora of cancers. Earlier studies from our group, have showcased that overexpression of KDM2B in the human prostate cancer cell line DU-145 is associated with cell adhesion, actin reorganization, and improved cancer cell migration. In addition, we have previously examined changes of cytosolic Ca2+, regulated by the pore-forming proteins ORAI and the Ca2+ sensing stromal interaction molecules (STIM), via store-operated Ca2+ entry (SOCE) in wild-type DU-145. This study sought to evaluate the impact of KDM2B overexpression on the expression of key molecules (SGK1, Nhe1, Orai1, Stim1) and SOCE. Furthermore, this is the first study to evaluate KDM2B expression in circulating tumor cells (CTCs) from patients with prostate cancer. mRNA levels for SGK1, Nhe1, Orai1, and Stim1 were quantified by RT-PCR. Calcium signals were measured in KDM2B-overexpressing DU-145 cells, loaded with Fura-2. Blood samples from 22 prostate cancer cases were scrutinized for KDM2B expression using immunofluorescence staining and the VyCAP system. KDM2B overexpression in DU-145 cells increased Orai1, Stim1, and Nhe1 mRNA levels and significantly decreased Ca2+ release. KDM2B expression was examined in 22 prostate cancer patients. CTCs were identified in 45 % of these patients. 80 % of the cytokeratin (CK)-positive patients and 63 % of the total examined CTCs exhibited the (CK + KDM2B + CD45−) phenotype. To conclude, this study is the first to report increased expression of KDM2B in CTCs from patients with prostate cancer, bridging in vitro and preclinical assessments on the potentially crucial role of KDM2B on migration, invasiveness, and ultimately metastasis in prostate cancer.

COVID-19 Diarrhea Is Inflammatory, Caused by Direct Viral Effects Plus Major Role of Virus-induced Cytokines

BACKGROUND AND AIMSDiarrhea occurs in up to 50% of cases of COVID-19. Nonetheless, the pathophysiologic mechanism(s) have not been determined. METHODSThis was examined using normal human enteroid monolayers exposed apically to live SARS-CoV-2 or non-replicating virus like particles (VLPs) bearing the four SARS-CoV-2 structural proteins or irradiated virus, all of which bound and entered enterocytes. RESULTSLive virus and VLPs increased secretion of multiple cytokines and reduced mRNAs of ACE2, NHE3 and DRA. IL-6 plus IL-8 alone reduced NHE3 mRNA and protein and DRA mRNA. Neither VLPs nor IL-6 plus IL-8 alone altered Cl- secretion, but together they caused Cl- secretion, which was Ca2+ dependent, CFTR independent, blocked partially by a specific TMEM16 A inhibitor, and entirely by a general TMEM16 family inhibitor. VLPs and irradiated virus, but not IL-6 plus IL-8, produced Ca2+ waves that began within minutes of VLP exposure, lasted for at least 60 min, and were prevented by pretreatment with apyrase; a P2Y1 receptor antagonist; and general TMEM16 family inhibitor but NOT by the specific TMEM16A inhibitor. CONCLUSIONSThe pathophysiology of COVID-19 diarrhea appears to be a unique example of a calcium dependent inflammatory diarrhea, that is caused by direct viral effects plus the virus-induced intestinal epithelial cytokine secretion.

IDH1 K224 acetylation promotes colorectal cancer via miR-9-5p/NHE1 axis-mediated regulation of acidic microenvironment

The acidic microenvironment is considered an important factor in colorectal cancer (CRC) that contributes to malignant transformation. However, the underlying mechanism remains unclear. In a previous study, we confirmed that IDH1 K224 deacetylation promotes enzymatic activity and the production of α-KG. Here, we further investigate the effect of IDH1 hyperacetylation on the CRC acidic microenvironment. We demonstrate that increased α-KG affects hydroxylation of Ago2 and mediates miR-9-5p targeting NHE1 protein. Knockdown of NHE1 dramatically attenuates CRC cell proliferation and migration by restricting transport of intracellular H+ out of cells. Furthermore, we show that miR-9-5p is the microRNA with the most significant difference in the alteration of IDH1 K224 acetylation and can downregulate NHE1 mRNA. Our data also indicate that hydroxylation stabilizes Ago2, which in turn promotes miR-9-5p activity. Taken together, our results reveal a novel mechanism through which IDH1 deacetylation regulates the cellular acidic microenvironment and inhibits CRC metastasis.

Altered colonocyte differentiation may lead to decreased electrolyte and fluid absorption in the ileocolon of IBD patients by affecting the expression pattern of the responsible ion transporters

Crohn's disease and ulcerative colitis are chronic inflammatory bowel diseases (IBD) characterized by disturbed homeostasis of the immune system, the intestinal mucosa and the microbiome, causing ‘leaky’ intestinal epithelial barrier with altered fluid and electrolyte absorption leading to diarrhea. Fluid absorption in the ileocolonic region is predominantly mediated by the Cl-/HCO3 - exchanger SLC26A3 (DRA) and the Na+/H+ exchanger SLC9A3 (NHE3), while in the distal colon, the epithelial Na+ channel (ENaC) also plays a role. The objective of the current study is to evaluate if the downregulation of specific absorptive ion transporters or general defect of the enterocyte differentiation correlate with the degree of diarrhea and inflammation. For this reason, we have analyzed the mRNA expression and/or protein abundance of DRA, NHE3 and ENaC, as well as a panel of genes that correlate with the differentiation state of the colonic epithelium, in different segments of the ileocolon of healthy individuals and IBD patients with an acute flare or in remission. Endoscopy and histopathologic scoring and TNFα mRNA levels were used to grade inflammation, clinical scores for disease activity and diarrhea. mRNA expression and immunohistochemical staining of DRA and NHE3 in healthy colonic tissue revealed differences in the expression pattern of DRA and NHE3 along the human colon, with DRA being most abundant in the transverse and descending colon and NHE3 in the ascending colon (n=10). The reduction of DRA and NHE3 mRNA expression/abundance in the membrane correlated with the degree of inflammation as well as the diarrhea score (n=42), while in inactive disease the expression and abundance of DRA and NHE3 did not differ from healthy controls (n=13). Furthermore, significant decrease of the ENaC subunit ENaCγ expression was detected in inflamed colon sigmoideum (n=30). Since the addressed ion transporters are expressed on differentiated intestinal epithelial cells, we further investigated if their altered expression is linked to a possible defect of the epithelial differentiation program. It was found that the mRNA expression of the absorptive enterocyte differentiation markers intestinal alkaline phosphatase (iALP), villin and HES1 was significantly decreased in inflamed colon, while the expression of the proliferative marker Ki67, HOPX, BM1 and Claudin2 was significantly increased. However, the expression of the stem cell marker LGR5 was not altered (n=30). The results showed a disrupted transcriptional regulation of the epithelial ion transporters DRA, NHE3 and ENaCγ in the inflamed epithelium of IBD patients, which will lead to their reduced function and cause electrolyte/water accumulation leading to diarrhea. The decreased expression of the epithelial ion transporters could be a consequence of the altered enterocyte differentiation of the inflamed epithelium, most likely due to signaling pathways triggered by the proinflamatory cytokines. Grant Support: DFG SE 460/21-1 and 22-1, and VW Z1953. This is the full abstract presented at the American Physiology Summit 2023 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.

Shikonin Alleviates Gentamicin-Induced Renal Injury in Rats by Targeting Renal Endocytosis, SIRT1/Nrf2/HO-1, TLR-4/NF-κB/MAPK, and PI3K/Akt Cascades.

Gentamicin causes kidney injury due to its accumulation in proximal tubule epithelial cells via the megalin/cubilin/CLC-5 complex. Recently, shikonin has been shown to have potential anti-inflammatory, antioxidant, antimicrobial, and chloride channel-inhibiting effects. The current study investigated the alleviation of gentamicin-induced renal injury by shikonin while preserving its bactericidal effect. Nine-week-old Wistar rats were administered 6.25, 12.5, and 25 mg/kg/day shikonin orally, one hour after the i.p. injection of 100 mg/kg/day gentamicin for seven days. Shikonin significantly and dose-dependently alleviated gentamicin-induced renal injury, as revealed by restoring normal kidney function and histological architecture. Furthermore, shikonin restored renal endocytic function, as indicated by suppressing the elevated renal megalin, cubilin, and CLC-5 and enhancing the reduced NHE3 levels and mRNA expressions induced by gentamicin. These potentials could be attributed to the modulation of the renal SIRT1/Nrf2/HO-1, TLR-4/NF-κB/MAPK, and PI3K/Akt cascades, which enhanced the renal antioxidant system and suppressed renal inflammation and apoptosis, as indicated by enhancements of SIRT1, Nrf2, HO-1, GSH, SOD, TAC, Iκb-α, Bcl-2, PI3K, and Akt levels and mRNA expressions, with reduction of TLR-4, NF-κB, MAPK, IL-1β, TNF-α, MDA, iNOS, NO, cytochrome c, caspase-3, Bax levels, and Bax/Bcl-2 ratio. Therefore, shikonin is a promising therapeutic agent for alleviating gentamicin-induced renal injury.

Cardiac Sodium/Hydrogen Exchanger (NHE11) as a Novel Potential Target for SGLT2i in Heart Failure: A Preliminary Study.

Despite the reduction of cardiovascular events, including the risk of death, associated with sodium/glucose cotransporter 2 inhibitors (SGLT2i), their basic action remains unclear. Sodium/hydrogen exchanger (NHE) has been proposed as the mechanism of action, but there are controversies related to its function and expression in heart failure (HF). We hypothesized that sodium transported-related molecules could be altered in HF and modulated through SGLT2i. Transcriptome alterations in genes involved in sodium transport in HF were investigated in human heart samples by RNA-sequencing. NHE11 and NHE1 protein levels were determined by ELISA; the effect of empagliflozin on NHE11 and NHE1 mRNA levels in rats’ left ventricular tissues was studied through RT-qPCR. We highlighted the overexpression of SLC9C2 and SCL9A1 sodium transport genes and the increase of the proteins that encode them (NHE11 and NHE1). NHE11 levels were correlated with left ventricular diameters, so we studied the effect of SGLT2i on its expression, observing that NHE11 mRNA levels were reduced in treated rats. We showed alterations in several sodium transports and reinforced the importance of these channels in HF progression. We described upregulation in NHE11 and NHE1, but only NHE11 correlated with human cardiac dysfunction, and its levels were reduced after treatment with empagliflozin. These results propose NHE11 as a potential target of SGLT2i in cardiac tissue.

Decreased NHE3 expression in colon cancer is associated with DNA damage, increased inflammation and tumor growth

Dysregulation of intra- and extracellular pH in cancer contributes to extracellular matrix remodeling, favors cell migration, proliferation, and metastasis. Although the primary attention has been focused on the role of the ubiquitous Na+/H+ exchanger isoform NHE1, the role of NHE3, the predominant apical isoform in colonic surface epithelium in the pathogenesis of colon cancer has not been investigated. Here, we show that NHE3 mRNA expression is significantly reduced in colorectal cancer patients and that low NHE3 expression is associated with poorer survival. Deletion of NHE3 in ApcMin mice evaluated at 15 weeks of age (significant mortality was observed beyond this time) led to lower body weights, increased mucosal inflammation, increased colonic tumor numbers, evidence of enhanced DNA damage in tumor surface epithelium, and to significant alteration in the gut microbiota. In the absence of the inflammatory and microbial pressors, ca. 70% knockdown of NHE3 expression in SK-CO15 cells led to reduced intracellular pH, elevated apical pH, dramatic differences in their transcriptomic profile, increased susceptibility to DNA damage, increased proliferation, decreased apoptosis and reduced adhesion to extracellular matrix proteins. Our findings suggest that loss of NHE3 in the surface epithelium of colonic tumors has profound consequences for cancer progression and behavior.

MiRNA-23a modulates sodium-hydrogen exchanger 1 expression: studies in medullary thick ascending limb of salt-induced hypertensive rats.

The kidney is the main organ in the pathophysiology of essential hypertension. Although most bicarbonate reabsorption occurs in the proximal tubule, the medullary thick ascending limb (mTAL) of the nephron also maintains acid-base balance by contributing to 25% of bicarbonate reabsorption. A crucial element in this regulation is the sodium-hydrogen exchanger 1 (NHE1), a ubiquitous membrane protein controlling intracellular pH, where proton extrusion is driven by the inward sodium flux. MicroRNA (miRNA) expression of hypertensive patients significantly differs from that of normotensive subjects. The aim of this study was to determine the functional role of miRNA alterations at the mTAL level. By miRNA microarray analysis, we identified miRNA expression profiles in isolated mTALs from high sodium intake-induced hypertensive rats (HSD) versus their normotensive counterparts (NSD). In vitro validation was carried out in rat mTAL cells. Five miRNAs involved in the onset of salt-sensitive hypertension were identified, including miR-23a, which was bioinformatically predicted to target NHE1 mRNA. Data demonstrated that miRNA-23a is downregulated in the mTAL of HSD rats while NHE1 is upregulated. Consistently, transfection of an miRNA-23a mimic in an mTAL cell line, using a viral vector, resulted in NHE1 downregulation. NHE1, a protein involved in sodium reabsorption at the mTAL level and blood pressure regulation, is upregulated in our model. This was due to a downregulation of miRNA-23a. Expression levels of this miRNA are influenced by high sodium intake in the mTALs of rats. The downregulation of miRNA-23a in humans affected by essential hypertension corroborate our data and point to the potential role of miRNA-23a in the regulation of mTAL function following high salt intake.

Effects of Netrin-1 and NHE1 Participation on the Migration of Macrophages Driven by CCL2.

This experiment was designed to investigate the relationship between NHE1 gene expression differences between Netrin-1 and NHE1. For this purpose, the blank control, CCL2, CCL2 + Netrin-1 groups were constructed, and cell migration ability was detected by scratch tests and Transwell experiments; Commercial over-expressed NHE1 adenovirus vector (over-expressed NHE1 group), shRNA adenoviral vector silencing NHE1 (silencing NHE1 group) and negative control without carrying virus (negative control group) were subjected to RT-PCR test 24h after infection and pH recovery rate after acid loading was measured. The percentage of wound healing area and the number of cell migration of macrophages in the blank control group, CCL2 group, CCL2+Netrin-1 group, over-expressed NHE1 group, silencing NHE1 group and negative control group were compared. Results showed that in terms of migration ability, the percentage of wound healing area and migration in CCL2 increased (P <0.05), in CCL2 + Netrin-1 (P <0.05) and increased NHE1 mRNA (P <0.05), and not in NHE1 (P <0.05).pH response rate after acid load (NHE1 activity) showed that NHE1 activity was enhanced compared with the blank group, while NHE1 activity in silent NHE1 group decreased (P <0.05); from macrophage migration ability after overexpression/silencing, the percentage of macrophage wound healing area and cell migration increased/decreased compared with CCL2 group and Netrin-1 + CCL2 group (P <0.05). Then Upregulation of NHE1 can promote CCL2-driven macrophage RAW264.7 cell migration, and the downregulation of NHE1 can inhibit its cell migration; Netrin-1 can inhibit CCL2-driven RAW264.7 cell migration regardless of NHE1 regulation.

Pulmonary endothelial cells from different vascular segments exhibit unique recovery from acidification and Na+/H+ exchanger isoform expression.

Sodium-hydrogen exchangers (NHEs) tightly regulate intracellular pH (pHi), proliferation, migration and cell volume. Heterogeneity exists between pulmonary endothelial cells derived from different vascular segments, yet the activity and isoform expression of NHEs between these vascular segments has not been fully examined. Utilizing the ammonium-prepulse and recovery from acidification technique in a buffer lacking bicarbonate, pulmonary microvascular and pulmonary artery endothelial cells exhibited unique recovery rates from the acid load dependent upon the concentration of the sodium transport inhibitor, amiloride; further, pulmonary artery endothelial cells required a higher dose of amiloride to inhibit sodium-dependent acid recovery compared to pulmonary microvascular endothelial cells, suggesting a unique complement of NHEs between the different endothelial cell types. While NHE1 has been described in pulmonary endothelial cells, all NHE isoforms have not been accounted for. To address NHE expression in endothelial cells, qPCR was performed. Using a two-gene normalization approach, Sdha and Ywhag were identified for qPCR normalization and analysis of NHE isoforms between pulmonary microvascular and pulmonary artery endothelial cells. NHE1 and NHE8 mRNA were equally expressed between the two cell types, but NHE5 expression was significantly higher in pulmonary microvascular versus pulmonary artery endothelial cells, which was confirmed at the protein level. Thus, pulmonary microvascular and pulmonary artery endothelial cells exhibit unique NHE isoform expression and have a unique response to acid load revealed through recovery from cellular acidification.

Pulmonary endothelial cells from different vascular compartments exhibit unique recovery from acidification and Na+/H+ exchanger isoform expression

Sodium‐hydrogen exchangers (NHEs) tightly regulate intracellular pH (pHi), proliferation, migration and cell volume. Heterogeneity exists between pulmonary endothelial cells derived from different vascular segments, yet the activity and isoform expression of NHEs between these vascular segments has not been fully examined.Utilizing the ammonium‐prepulse and recovery from acidification technique in a buffer lacking bicarbonate, pulmonary microvascular and pulmonary artery endothelial cells exhibited a similar rate of recovery from the acid load regardless of the concentrations of the sodium transport inhibitor, amiloride; however, pulmonary artery endothelial cells required a higher dose of amiloride to inhibit sodium‐dependent acid recovery compared to pulmonary microvascular endothelial cells, suggesting a unique complement of NHEs between the different endothelial cell types. While NHE1 has been described in pulmonary endothelial cells, all NHE isoforms have not been accounted for. To address NHE expression in endothelial cells, qPCR was performed. Using a two‐gene normalization approach, Sdha and Ywhag were identified for qPCR normalization and analysis of NHE isoforms between pulmonary microvascular and pulmonary artery endothelial cells. NHE1 and NHE8 mRNA were equally expressed between the two cell types, but NHE5 expression was significantly higher in pulmonary microvascular and pulmonary artery endothelial cells.Thus, pulmonary microvascular and pulmonary artery endothelial cells exhibit unique NHE isoform expression and have a unique response to acid load revealed through recovery from cellular acidification.

Rosmarinic Acid, as an NHE1 Activator, Decreases Skin Surface pH and Improves the Skin Barrier Function.

Stratum corneum (SC) pH regulates skin barrier functions and elevated SC pH is an important factor in various inflammatory skin diseases. Acidic topical formulas have emerged as treatments for impaired skin barriers. Sodium proton exchanger 1 (NHE1) is an important factor in SC acidification. We investigated whether topical applications containing an NHE1 activator could improve skin barrier functions. We screened plant extracts to identify NHE1 activators in vitro and found Melissa officinalis leaf extract. Rosmarinic acid, a component of Melissa officinalis leaf extract, significantly increased NHE1 mRNA expression levels and NHE1 production. Immunofluorescence staining of NHE1 in 3D-cultured skin revealed greater upregulation of NHE1 expression by NHE1 activator cream, compared to vehicle cream. Epidermal lipid analysis revealed that the ceramide level was significantly higher upon application of the NHE1 activator cream on 3D-cultured skin, compared to application of a vehicle cream. In a clinical study of 50–60-year-old adult females (n = 21), application of the NHE1 activator-containing cream significantly improved skin barrier functions by reducing skin surface pH and transepidermal water loss and increasing skin hydration, compared to patients who applied vehicle cream and those receiving no treatment. Thus, creams containing NHE1 activators, such as rosmarinic acid, could help maintain or recover skin barrier functions.

Sodium/hydrogen-exchanger-2 modulates colonocyte lineage differentiation.

The sodium/hydrogen exchanger 2 (NHE2) is an intestinal acid extruder with crypt-predominant localization and unresolved physiological significance. Our aim was to decipher its role in colonic epithelial cell proliferation, differentiation and electrolyte transport. Alterations induced by NHE2-deficiency were addressed in murine nhe2-/- and nhe2+/+ colonic crypts and colonoids, and NHE2-knockdown and control Caco2Bbe cells using pH-fluorometry, gene expression analysis and immunofluorescence. pHi -measurements along the colonic cryptal axis revealed significantly decreased intracellular pH (pHi ) in the middle segment of nhe2-/- compared to nhe2+/+ crypts. Increased Nhe2 mRNA expression was detected in murine colonoids in the transiently amplifying/progenitor cell stage (TA/PE). Lack of Nhe2 altered the differentiation programme of colonic epithelial cells with reduced expression of absorptive lineage markers alkaline phosphatase (iAlp), Slc26a3 and transcription factor hairy and enhancer-of-split 1 (Hes1), but increased expression of secretory lineage markers Mucin 2, trefoil factor 3 (Tff3), enteroendocrine marker chromogranin A and murine atonal homolog 1 (Math1). Enterocyte differentiation was found to be pHi dependent with acidic pHi reducing, and alkaline pHi stimulating the expression of enterocyte differentiation markers in Caco2Bbe cells. A thicker mucus layer, longer crypts and an expanded brush border membrane zone of sodium/hydrogen exchanger 3 (NHE3) abundance may explain the lack of inflammation and the normal fluid absorptive rate in nhe2-/- colon. The results suggest that NHE2 expression is activated when colonocytes emerge from the stem cell niche. Its activity increases progenitor cell pHi and thereby supports absorptive enterocyte differentiation.

Estrogen-related receptor γ2 controls NaCl uptake to maintain ionic homeostasis.

Estrogen-related receptors (ERRs) are known to function in mammalian kidney as key regulators of ion transport-related genes; however, a comprehensive understanding of the physiological functions of ERRs in vertebrate body fluid ionic homeostasis is still elusive. Here, we used medaka (Oryzias melastigma), a euryhaline teleost, to investigate how ERRs are involved in ion regulation. After transferring medaka from hypertonic seawater to hypotonic freshwater (FW), the mRNA expression levels of errγ2 were highly upregulated, suggesting that Errγ2 may play a crucial role in ion uptake. In situ hybridization showed that errγ2 was specifically expressed in ionocytes, the cells responsible for Na+/Cl- transport. In normal FW, ERRγ2 morpholino knockdown caused reductions in the mRNA expression of Na+/Cl- cotransporter (Ncc), the number of Ncc ionocytes, Na+/Cl- influxes of ionocytes, and whole-body Na+/Cl- contents. In FW with low Na+ and low Cl-, the expression levels of mRNA for Na+/H+ exchanger 3 (Nhe3) and Ncc were both decreased in Errγ2 morphants. Treating embryos with DY131, an agonist of Errγ, increased the whole-body Na+/Cl- contents and ncc mRNA expression in Errγ2 morphants. As such, medaka Errγ2 may control Na+/Cl- uptake by regulating ncc and/or nhe3 mRNA expression and ionocyte number, and these regulatory actions may be subtly adjusted depending on internal and external ion concentrations. These findings not only provide new insights into the underpinning mechanism of actions of ERRs, but also enhance our understanding of their roles in body fluid ionic homeostasis for adaptation to changing environments during vertebrate evolution.

Functional characterization of the sodium/hydrogen exchanger 8 and its role in proliferation of colonic epithelial cells.

Intestinal NaCl, HCO3-, and fluid absorption are strongly dependent on apical Na+/H+ exchange. The intestine expresses three presumably apical sodium-hydrogen exchanger (NHE) isoforms: NHE2, NHE3, and NHE8. We addressed the role of NHE8 [solute carrier 9A8 (SLC9A8)] and its interplay with NHE2 (SLC9A2) in luminal proton extrusion during acute and chronic enterocyte acidosis and studied the differential effects of NHE8 and NHE2 on enterocyte proliferation. In contrast to NHE3, which was upregulated in differentiated versus undifferentiated colonoids, the expression of NHE2 and NHE8 remained constant during differentiation of colonoids and Caco2Bbe cells. Heterogeneously expressed Flag-tagged rat (r)Nhe8 and human (h)NHE8 translocated to the apical membrane of Caco2Bbe cells. rNhe8 and hNHE8, when expressed in NHE-deficient PS120 fibroblasts showed higher sensitivity to HOE642 compared to NHE2. Lentiviral shRNA knockdown of endogenous NHE2 in Caco2Bbe cells (C2Bbe/shNHE2) resulted in a decreased steady-state intracellular pH (pHi), an increased NHE8 mRNA expression, and augmented NHE8-mediated apical NHE activity. Lentiviral shRNA knockdown of endogenous NHE8 in Caco2Bbe cells (C2Bbe/shNHE8) resulted in a decreased steady-state pHi as well, accompanied by decreased NHE2 mRNA expression and activity, which together contributed to reduced apical NHE activity in the NHE8-knockdown cells. Chronic acidosis increased NHE8 but not NHE2 mRNA expression. Alterations in NHE2 and NHE8 expression/activity affected proliferation, with C2Bbe/shNHE2 cells having lower and C2Bbe/shNHE8 having higher proliferative capacity, accompanied by amplified ERK1/2 signaling pathway and increased EGFR expression in the latter cell line. Thus, both Na+/H+ exchangers have distinct functions during cellular homeostasis by triggering different signaling pathways to regulate cellular proliferation and pHi control.

Dysregulated ion channels and transporters activate endoplasmic reticulum stress in rat kidney of fetal growth restriction

AimsThe mechanisms underlying the fetal origin of renal disease remains unknown. This study aimed to investigate the profiles of ion channel and transporter proteins in the fetal kidney in fetal growth restriction (FGR)rats, and to explore their association with the fetal origin of renal disease. Main methodsAn FGR rat model was developed by administration of a low-protein diet. Then 367 differentially expressed proteins (DEPs) from quantitative proteome analysis were subjected to Ingenuity Pathway Analysis. 22 DEPs associated with ion channels/transporters were evaluated in the fetal kidney. Na+/H+ exchanger1(NHE1) and its downstream unfolded protein response (UPR) pathway were investigated. Furthermore, overexpression of NHE1 were achieved via plasmid transfection to evaluate the potential influence on the UPR pathway and cell apoptosis in human proximal tubular epithelial cell line HK2 cells. Key findingsFindings were as follows: 1) In the FGR fetal kidney, aquaporin 2/4, solute carrier (SLC) 8a1, 33a1, etc. were downregulated, whereas other transporters including SLC 2a1, 4a1, 9a1, 29a3, etc. were upregulated. 2) NHE1 mRNA levels were markedly elevated in the FGR fetus. Further investigation revealed an increase in the UPR pathway regulators. 3) In vitro study showed that NHE1 overexpression in HK2 cells significantly induced expression of the endoplasmic reticulum stress (ERS) regulators and led to a decrease in the anti-apoptotic potential. SignificanceWe speculate that maternal protein malnutrition causes dysregulation of ion channels/transporters in the fetal kidney. Upregulated NHE1 may activate the UPR pathway and induce cell apoptosis thus leading to impairment of kidney function.

Generation and Characterization of Thick Ascending Limb‐Specific NHE3 Knockout Mice

The Na+/H+ exchanger 3 (NHE3) mediates Na+ absorption in the intestine and reabsorption in the kidney, thereby playing a key role in fluid homeostasis and blood pressure regulation. NHE3 is expressed in the luminal membrane of the renal proximal tubule (PT), where the majority of renal Na+ reabsorption (~67%) occurs. In addition, NHE3 is expressed substantially in the thick ascending limb (TAL). Whole body NHE3 knockout mice exhibit hypovolemia, hypotension, diarrhea, metabolic acidosis and renal Na+ wasting. Studies in kidney‐specific (PT and TAL) NHE3 knockout mice showed a rather mild phenotype in terms of Na+ homeostasis and no acid‐base phenotype (Kidney International; 2017,92:397–414). To determine the physiological role of NHE3 in the TAL we generated a novel tamoxifen (Tam)‐inducible TAL‐specific NHE3 knockout (NHE3TAL‐KO) mouse model by crossing NHE3loxlox (Control, Con) with uromodulin Cre (UmodCreERT2) mice. After Tam administration (3 days, 67 mg/kg via oral gavage), TAL‐specific NHE3 knockout was confirmed by quantitative RT‐qPCR for NHE3 in microdissected inner medulla/inner stripe outer medulla (ISOM) and renal cortex and by immunofluorescent labeling with NHE3 (PT and TAL), NKCC2 (TAL marker) and lotus tetragonolobus lectin (LTL, PT marker). NHE3 mRNA levels in NHE3TAL‐KO mice (n=8) were reduced by 30% in the renal cortex and by 94% in the ISOM. Control mice showed colocalization of NHE3 with LTL and NKCC2; in contrast, no immunofluorescence was observed for NHE3 in the TAL of NHE3TAL‐KO mice. NHE3TAL‐KO mice were born at predicted Mendelian frequencies, appeared grossly indistinguishable from Con mice, and developed normally. Two weeks after Tam administration, NHE3TAL‐KO mice (n=11) showed a ~25% lower urine osmolality compared to Con mice (n=10) (2224±90 versus 1826±93 mOsm/kg, P&lt;0.05) associated with ~20% higher water intake (5.3±0.2 versus 6.3±0.3 mL/day, P&lt;0.05). Blood pH was slightly but significantly increased in NHE3TAL‐KO mice (n=20) versus Con mice (n=19) (7.39±0.01 versus 7.37±0.01, P&lt;0.05), which was associated with a tendency of higher blood HCO3− in NHE3TAL‐KO mice (26.1±0.4 versus 25.5±0.3 mmol, P=0.07). However, no differences were observed for urinary pH (6.1±0.1 vs 6.2±0.1, NS), urinary Na+/creatinine (37±5 vs 41±5 mmol/mmol, NS), K+/creatinine (81±6 vs 94±7 mmol/mmol, NS), plasma Na+ (147±1 versus 147±1 mmol, NS) or plasma K+ (4.2±0.1 vs 4.4±0.1 mmol, NS). In conclusion, we successfully generated TAL‐specific NHE3 knockout mouse and found that NHE3 in the TAL contributes to urinary concentrating ability and acid‐base regulation. This mouse model will further be useful to delineate the TAL‐specific role of NHE3 for renal function.Support or Funding InformationTR is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK110621) and American Heart Association Transformational Research Award (19TPA34850116), LT was supported by an American Heart Association Postdoctoral Fellowship (19POST34400026) and JX was supported by an American Heart Association Predoctoral Fellowship (18PRE33990236).

Proximal tubule albumin uptake – potential role for endothelin system in sickle cell disease mice

Elevated plasma endothelin‐1 (ET‐1) reported in sickle cell disease (SCD) patients correlate with microalbuminuria, a major mortality risk factor in SCD. ET‐1 contributes to glomerular and tubular injury in SCD, as evidenced by increased glomerular permeability to albumin and urinary excretion of tubular injury markers. Although selective ETA receptor antagonism reduces albuminuria in humanized sickle cell (HbSS) mice, the mechanism of this action remains unclear. The aim of the study was to determine whether selective ETA receptor antagonism prevents albuminuria by preserving the expression of tubular albumin‐associated transporters in proximal tubule (PT) cells. Male C57BL/6 or HbSS and genetic control (HbAA) mice were used to determine the effect of ET‐1 on the expression of proximal tubule albumin trafficking mediators, such as megalin and NHE‐3. Exposure of primary mouse PT cells to ET‐1 (50nM) for 8h decreased megalin (53% reduction) and doubled NHE‐3 expression. Pre‐treatment with the ETA antagonist, BQ123 (1 μM), preserved megalin expression and had no effect on NHE‐3. Moreover, selective ETB receptor blockade (BQ788, 1 μM) prevented ET‐1‐mediated increase in NHE‐3 expression. Primary PT cells isolated from HbSS mice showed decreased megalin mRNA expression as well as protein abundance relative to HbAA PT controls. Ten‐week treatment with selective ETA receptor antagonist (10mg/kg/day) preserved expression of megalin at control levels. There were no differences in NHE‐3 mRNA expression in HbSS PT cells regardless the treatment. Interestingly, PT cells from HbSS cultured with HbSS plasma and ET‐1 (10nM) had decreased NHE‐3 protein abundance compared to non‐treated cells. These results potentially uncover a novel role for ET‐1 in PT albumin handling and suggest that PT ET‐1 receptor signaling contributes to albuminuria in SCD.Support or Funding InformationSupport provided by an NIH U01 HL117684 to D.M.P, AHA 19CDA34660073 and NIH‐NHLBI K99HL144817 to M.K.

Impairment of electroneutral Na+transport and associated downregulation of NHE3 contributes to the development of diarrhea following in vivo challenge withBrachyspiraspp.

The effect of Brachyspira hyodysenteriae and Brachyspira hampsonii spirochetosis on Na+ transport was assessed in the colon to determine its contribution to diarrheal disease in pigs following experimental infection. Electrogenic and electroneutral Na+ absorption was assessed in Ussing chambers by radiolabeled 22Na flux and pharmacological inhibitory studies. Basal radiolabeled 22Na flux experiments revealed that mucosal-to-serosal flux (Jms) was significantly impaired in B. hyodysenteriae and B. hampsonii-diseased pigs. Inhibition of epithelial sodium channel via amiloride did not significantly reduce electrogenic short-circuit current (Isc) in the proximal, apex, and distal colonic segments of diseased pigs over control pigs, suggesting that a loss of electroneutral Na+ absorption is responsible for diarrheal development. These findings were further supported by significant downregulation of Na+/H+ exchanger (NHE1, NHE2, and NHE3) mRNA expression in the proximal, apex, and distal colonic segments paired with decreased protein expression of the critical NHE3 isoform. The decrease in NHE3 mRNA expression appears not to be attributed to the host's cytokine response as human IL-1α did not modify NHE3 mRNA expression in Caco-2 cells. However, a whole cell B. hampsonii lysate significantly downregulated NHE3 mRNA expression and significantly increased p38 phosphorylation in Caco-2 cells. Together these findings provide a likely mechanism for the spirochete-induced malabsorptive diarrhea, indicated by a decrease in electroneutral Na+ absorption in the porcine colon due to Brachyspira's ability to inhibit NHE3 transcription, resulting in diarrheal disease.NEW & NOTEWORTHY This research demonstrates that diarrheal disease caused by two infectious spirochete spp. is a result of impaired electroneutral Na+ absorption via Na+/H+ exchanger 3 (NHE3) in the porcine colon. Our findings suggest that the decrease in NHE3 mRNA and protein is not likely a result of the host's cytokine response. Rather, it appears that these two Brachyspira spp. directly inhibit the transcription and translation of NHE3, resulting in the development of diarrhea.