Intestinal NaCl transport in NHE2 and NHE3 knockout mice

2002 ◽  
Vol 282 (5) ◽  
pp. G776-G784 ◽  
Author(s):  
Lara R. Gawenis ◽  
Xavier Stien ◽  
Gary E. Shull ◽  
Patrick J. Schultheis ◽  
Alison L. Woo ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Intestinal Tract ◽  
Apical Membrane ◽  
Intracellular Camp ◽  
Wild Type ◽  
Rt Pcr ◽  
Nacl Transport ◽  
Nacl Absorption ◽  
Small Intestinal ◽  
Absorptive Flux

Sodium/proton exchangers [Na+/H+ (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na+ and Cl− flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(−)], and NHE3 knockout [NHE3(−)] mice. Under basal conditions, NHE(+) and NHE2(−) jejuna had similar rates of net Na+ (∼6 μeq/cm2 · h) and Cl− (∼3 μeq/cm2 · h) absorption. In contrast, NHE3(−) jejuna had reduced net Na+ absorption (∼2 μeq/cm2 · h) but absorbed Cl− at rates similar to NHE(+) and NHE2(−) jejuna. Treatment with 100 μM 5-( N-ethyl- N-isopropyl) amiloride (EIPA) completely inhibited net Na+ and Cl−absorption in all genotypes. Studies of the Na+ absorptive flux ( J [Formula: see text]) indicated that J [Formula: see text] in NHE(+) jejunum was not sensitive to 1 μM EIPA, whereas J [Formula: see text] in NHE3(−) jejunum was equally sensitive to 1 and 100 μM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMPi) abolished net NaCl absorption and stimulated electrogenic Cl− secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(−) and NHE3(−) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na+ absorption; 2) an amiloride-sensitive Na+ transporter partially compensates for Na+ absorption in NHE3(−) jejunum; 3) cAMPi stimulation abolishes net Na+ absorption in NHE(+), NHE2(−), and NHE3(−) jejunum; and 4) electroneutral Cl− absorption is not directly dependent on either NHE2 or NHE3.

scholarly journals NADPH oxidase 4 mediates flow-induced superoxide production in thick ascending limbs

2012 ◽  
Vol 303 (8) ◽  
pp. F1151-F1156 ◽  
Author(s):  
Nancy J. Hong ◽  
Jeffrey L. Garvin
Keyword(s):  
Nadph Oxidase ◽  
Knockout Mice ◽  
Thick Ascending Limb ◽  
Wild Type ◽  
Rt Pcr ◽  
Nacl Transport ◽  
Flow Rates ◽  
Physiological Flow ◽  
Nadph Oxidase 4 ◽  
Luminal Flow

We previously showed that luminal flow stimulates thick ascending limb (TAL) superoxide (O2−) production by stretching epithelial cells and increasing NaCl transport, and reported that the major source of flow-induced O2− is NADPH oxidase (Nox). However, the specific Nox isoform involved is unknown. Of the three isoforms expressed in the kidney—Nox1, Nox2, and Nox4—we hypothesized that Nox4 is responsible for flow-induced O2− production in TALs. Measurable flow-induced O2− production at physiological flow rates of 0, 5, 10, and 20 nl/min was 5 ± 1, 9 ± 2, 36 ± 6, and 66 ± 8 AU/s, respectively. RT-PCR detected mRNA for all three Nox isoforms in the TAL. The order of RNA abundance was Nox2 > Nox4 >>> Nox1. Since all three isoforms are expressed in TALs and pharmacological inhibitors are not selective, we used rats transduced with siRNA and knockout mice. Nox4 siRNA knocked down Nox4 mRNA expression by 63 ± 7% but did not reduce Nox1 or Nox2 mRNA. Flow-induced O2− was 18 ± 9 AU/s in TALs transduced with Nox4 siRNA compared with 77 ± 9 AU/s in tubules transduced with scrambled siRNA. Flow-induced O2− was 81 ± 5 AU/s in Nox2 knockout mice compared with 83 ± 13 AU/s in wild-type mice. In TALs transduced with Nox1 siRNA, flow-induced O2− was 82 ± 7 AU/s. We conclude that Nox4 mediates flow-induced O2− production in TALs.

PAT-1 (Slc26a6) is the predominant apical membrane Cl−/HCO3− exchanger in the upper villous epithelium of the murine duodenum

2007 ◽  
Vol 292 (4) ◽  
pp. G1079-G1088 ◽  
Author(s):  
Janet E. Simpson ◽  
Clifford W. Schweinfest ◽  
Gary E. Shull ◽  
Lara R. Gawenis ◽  
Nancy M. Walker ◽  
...  
Keyword(s):  
Anion Exchanger ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Duodenal Mucosa ◽  
Transport Processes ◽  
Wild Type ◽  
Rt Pcr ◽  
Targeted Deletion ◽  
Anion Transporter ◽  
Dependent Transport

Basal HCO3− secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl−/HCO3− exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl−/HCO3− exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl−/HCO3− exchange activity was reduced by 65–80% in the PAT-1(−) duodenum, 30–40% in the DRA(−) duodenum, and <5% in the AE4(−) duodenum compared with the WT duodenum. SO42−/HCO3− exchange was eliminated in the PAT-1(−) duodenum but was not affected in the DRA(−) and AE4(−) duodenum relative to the WT duodenum. Intracellular pH (pHi) was reduced in the PAT-1(−) villous epithelium but increased to WT levels in the absence of CO2/HCO3− or during methazolamide treatment. Further experiments under physiological conditions indicated active pHi compensation in the PAT-1(−) villous epithelium by combined activities of Na+/H+ exchanger 1 and Cl−-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl−/HCO3− and SO42−/HCO3− exchange across the apical membrane and 2) PAT-1 plays a role in pHi regulation in the upper villous epithelium of the murine duodenum.

Effect of Slc26a6 deletion on apical Cl−/HCO3− exchanger activity and cAMP-stimulated bicarbonate secretion in pancreatic duct

2007 ◽  
Vol 292 (1) ◽  
pp. G447-G455 ◽  
Author(s):  
Hiroshi Ishiguro ◽  
Wan Namkung ◽  
Akiko Yamamoto ◽  
Zhaohui Wang ◽  
Roger T. Worrell ◽  
...  
Keyword(s):  
Pancreatic Duct ◽  
Apical Membrane ◽  
Pancreatic Ducts ◽  
Bicarbonate Secretion ◽  
Wild Type ◽  
Rt Pcr ◽  
Secretory Rate ◽  
Transport Fluid ◽  
Exchange Activity

The role of Slc26a6 (PAT1) on apical Cl−/HCO3− exchange and bicarbonate secretion in pancreatic duct cells was investigated using Slc26a6 null and wild-type (WT) mice. Apical Cl−/HCO3− exchange activity was measured with the pH-sensitive dye BCECF in microperfused interlobular ducts. The HCO3−-influx mode of apical [Cl−]i/[HCO3−]o exchange (where brackets denote concentration and subscripts i and o denote intra- and extracellular, respectively) was dramatically upregulated in Slc26a6 null mice ( P < 0.01 vs. WT), whereas the HCO3−-efflux mode of apical [Cl−]o/[HCO3−]i exchange was decreased in Slc26a6 null mice ( P < 0.05 vs. WT), suggesting the unidirectionality of the Slc26a6-mediated HCO3− transport. Fluid secretory rate in interlobular ducts were comparable in WT and Slc26a6 null mice ( P > 0.05). In addition, when pancreatic juice was collected from whole animal in basal and secretin-stimulated conditions, neither juice volume nor its pH showed differences between WT and Slc26a6 null mice. Semiquantitative RT-PCR demonstrated more than fivefold upregulation in Slc26a3 (DRA) expression in Slc26a6 knockout pancreas. In conclusion, these results point to the role of Slc26a6 in HCO3− efflux at the apical membrane and also suggest the presence of a robust Slc26a3 compensatory upregulation, which can replace the function of Slc26a6 in pancreatic ducts.

scholarly journals Analysis of uterine gene expression in interleukin-15 knockout mice reveals uterine natural killer cells do not play a major role in decidualization and associated angiogenesis

Reproduction ◽  
2012 ◽  
Vol 143 (3) ◽  
pp. 359-375 ◽  
Author(s):  
Brent M Bany ◽  
Charles A Scott ◽  
Kirsten S Eckstrum
Keyword(s):  
Gene Expression ◽  
Natural Killer ◽  
Differential Expression ◽  
Knockout Mice ◽  
Implantation Site ◽  
Wild Type ◽  
Rt Pcr ◽  
Unk Cells ◽  
Interleukin 15 ◽  
Uterine Natural Killer

During decidualization, uterine natural killer (uNK) cells are the most abundant immune cell types found in the uterus. Although it is well known that they play key roles in spiral arteriole modification and the maintenance of decidual integrity seen after mid-pregnancy, their roles in the differentiation of decidual cells and accompanying angiogenesis during the process of decidualization is less well characterized. To address this, we used whole-genome Illumina BeadChip analysis to compare the gene expression profiles in implantation segments of the uterus during decidualization on day 7.5 of pregnancy between wild-type and uNK cell-deficient (interleukin-15-knockout) mice. We found almost 300 differentially expressed genes and verified the differential expression of ∼60 using quantitative RT-PCR. Notably, there was a lack of differential expression of genes involved in decidualization and angiogenesis and this was also verified by quantitative RT-PCR. Similar endothelial cell densities and proliferation indices were also found in the endometrium between the implantation site tissues of wild-type and knockout mice undergoing decidualization. Overall, the results of this study reveal that uNK cells likely do not play a major role in decidualization and accompanying angiogenesis during implantation. In addition, the study identifies a large number of genes whose expression in implantation-site uterine tissue during decidualization depends on interleukin-15 expression in mice.

What is the role of p53 during the cyst formation of Trichinella spiralis? A comparable study between knockout mice and wild type mice

Parasitology ◽  
2005 ◽  
Vol 131 (5) ◽  
pp. 705-712 ◽  
Author(s):  
T. BOONMARS ◽  
Z. WU ◽  
I. NAGANO ◽  
Y. TAKAHASHI
Keyword(s):  
Knockout Mice ◽  
Nurse Cell ◽  
Kinase Inhibitor ◽  
Trichinella Spiralis ◽  
Cell Formation ◽  
Cyst Formation ◽  
Wild Type ◽  
Rt Pcr ◽  
P53 Expression ◽  
Cyclin Dependent Kinase Inhibitor

During the cyst formation of Trichinella spiralis, the infected muscle cell undergoes basophilic change and apoptosis, which results in nurse cell formation. This study revealed expression kinetics of some apoptosis genes such as p53 and its closely related genes (tumor suppressor genes p53, p53; mouse double minute 2, MDM2; cyclin-dependent kinase inhibitor p21, p21waf). RT-PCR (reverse transcription polymerase chain reaction) results showed that these genes were temporarily expressed in the infected muscles during the cyst formation period, but not in normal muscles (or very low if any), which suggested the involvement of these apoptosis genes in the nurse cell formation. Cysts and neighbouring muscle cells were separately collected and RT-PCR was performed, which suggested that p53 was expressed in the cysts. An immunocytochemical study showed that p53 was expressed in the nucleoplasm of basophilic cell in the cyst and Trichinella larvae, which suggested involvement of these apoptosis genes in the nurse cell formation. The same p53 expression kinetic study was performed on p53 knockout mice. The knockout mice did not express p53 genes, but expressed the other apoptosis genes in the same kinetics with only minor exceptions, suggesting that the expressions of these genes during the cyst formation were more or less p53-independent. There were no differences in the number and morphology of the cysts between the knockout mice and wild type mice. Thus apoptosis seen during the Trichinella cyst formation can be operated in the presence or absence of p53.

Somatostatin stimulates ductal bile absorption and inhibits ductal bile secretion in mice via SSTR2 on cholangiocytes

2003 ◽  
Vol 284 (5) ◽  
pp. C1205-C1214 ◽  
Author(s):  
Ai-Yu Gong ◽  
Pamela S. Tietz ◽  
Melissa A. Muff ◽  
Patrick L. Splinter ◽  
Robert C. Huebert ◽  
...  
Keyword(s):  
Bile Duct ◽  
Knockout Mice ◽  
Somatostatin Receptor ◽  
Fluid Secretion ◽  
Intracellular Camp ◽  
Fluid Absorption ◽  
Wild Type ◽  
Bile Formation ◽  
Luminal Area ◽  
Ductal Fluid

With an in vitro model using enclosed intrahepatic bile duct units (IBDUs) isolated from wild-type and somatostatin receptor (SSTR) subtype 2 knockout mice, we tested the effects of somatostatin, secretin, and a selective SSTR2 agonist (L-779976) on fluid movement across the bile duct epithelial cell layer. By RT-PCR, four of five known subtypes of SSTRs (SSTR1, SSTR2A/2B, SSTR3, and SSTR4, but not SSTR5) were detected in cholangiocytes in wild-type mice. In contrast, SSTR2A/2B were completely depleted in the SSTR2 knockout mice whereas SSTR1, SSTR3 and SSTR4 were expressed in these cholangiocytes. Somatostatin induced a decrease of luminal area of IBDUs isolated from wild-type mice, reflecting net fluid absorption; L-779976 also induced a comparable decrease of luminal area. No significant decrease of luminal area by either somatostatin or L-779976 was observed in IBDUs from SSTR2 knockout mice. Secretin, a choleretic hormone, induced a significant increase of luminal area of IBDUs of wild-type mice, reflecting net fluid secretion; somatostatin and L-779976 inhibited ( P< 0.01) secretin-induced fluid secretion. The inhibitory effect of both somatostatin and L-779976 on secretin-induced IBDU secretion was absent in IBDUs of SSTR2 knockout mice. Somatostatin induced an increase of intracellular cGMP and inhibited secretin-stimulated cAMP synthesis in cholangiocytes; depletion of SSTR2 blocked these effects of somatostatin. These data suggest that somatostatin regulates ductal bile formation in mice not only by inhibition of ductal fluid secretion but also by stimulation of ductal fluid absorption via interacting with SSTR2 on cholangiocytes, a process involving the intracellular cAMP/cGMP second messengers.

Novel Schering and ouabain-insensitive potassium-dependent proton secretion in the mouse cortical collecting duct

2002 ◽  
Vol 282 (1) ◽  
pp. F133-F143 ◽  
Author(s):  
Snezana Petrovic ◽  
Zachary Spicer ◽  
Tracey Greeley ◽  
Gary E Shull ◽  
Manoocher Soleimani
Keyword(s):  
Knockout Mice ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Wild Type ◽  
Acid Base ◽  
Proton Secretion ◽  
Relative Contribution ◽  
Collecting Tubule ◽  
Cortical Collecting Tubule

The intercalated (IC) cells of the cortical collecting duct (CCD) are important to acid-base homeostasis by secreting acid and reabsorbing bicarbonate. Acid secretion is mediated predominantly by apical membrane Schering (SCH-28080)-sensitive H+-K+- ATPase (HKA) and bafilomycin-sensitive H+-ATPase. The SCH-28080-sensitive HKA is believed to be the gastric HKA (HKAg). Here we examined apical membrane potassium-dependent proton secretion in IC cells of wild-type HKAg (+/+) and HKAg knockout (−/−) mice to determine relative contribution of HKAg to luminal proton secretion. The results demonstrated that HKAg (−/−) and wild-type mice had comparable rates of potassium-dependent proton secretion, with HKAg (−/−) mice having 100% of K+-dependent H+ secretion vs. wild-type mice. Potassium-dependent proton secretion was resistant to ouabain and SCH-28080 in HKAg knockout mice but was sensitive to SCH-28080 in wild-type animals. Northern hybridizations did not demonstrate any upregulation of colonic HKA in HKAg knockout mice. These data indicate the presence of a previously unrecognized K+-dependent SCH-28080 and ouabain-insensitive proton secretory mechanism in the cortical collecting tubule that may play an important role in acid-base homeostasis.

scholarly journals Constitutive depletion of Slc34a2/NaPi-IIb in rats causes perinatal mortality

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Eva Maria Pastor-Arroyo ◽  
Josep M. Monné Rodriguez ◽  
Giovanni Pellegrini ◽  
Carla Bettoni ◽  
Moshe Levi ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Intestinal Absorption ◽  
Intestinal Tract ◽  
Apical Membrane ◽  
Wild Type ◽  
Intestinal Epithelia ◽  
Dietary Phosphate ◽  
Perinatal Lethality ◽  
Paracellular Pathways ◽  
Inactivating Mutations

AbstractAbsorption of dietary phosphate (Pi) across intestinal epithelia is a regulated process mediated by transcellular and paracellular pathways. Although hyperphosphatemia is a risk factor for the development of cardiovascular disease, the amount of ingested Pi in a typical Western diet is above physiological needs. While blocking intestinal absorption has been suggested as a therapeutic approach to prevent hyperphosphatemia, a complete picture regarding the identity and regulation of the mechanism(s) responsible for intestinal absorption of Pi is missing. The Na+/Pi cotransporter NaPi-IIb is a secondary active transporter encoded by the Slc34a2 gene. This transporter has a wide tissue distribution and within the intestinal tract is located at the apical membrane of epithelial cells. Based on mouse models deficient in NaPi-IIb, this cotransporter is assumed to mediate the bulk of active intestinal absorption of Pi. However, whether or not this is also applicable to humans is unknown, since human patients with inactivating mutations in SLC34A2 have not been reported to suffer from Pi depletion. Thus, mice may not be the most appropriate experimental model for the translation of intestinal Pi handling to humans. Here, we describe the generation of a rat model with Crispr/Cas-driven constitutive depletion of Slc34a2. Slc34a2 heterozygous rats were indistinguishable from wild type animals under standard dietary conditions as well as upon 3 days feeding on low Pi. However, unlike in humans, homozygosity resulted in perinatal lethality.

Colonic H+-K+-ATPase in K+conservation and electrogenic Na+ absorption during Na+ restriction

2001 ◽  
Vol 281 (6) ◽  
pp. G1369-G1377 ◽  
Author(s):  
Zachary Spicer ◽  
Lane L. Clarke ◽  
Lara R. Gawenis ◽  
Gary E. Shull
Keyword(s):  
Knockout Mice ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Wild Type ◽  
Restricted Diet ◽  
Serum Aldosterone

Upregulation of the colonic H+-K+- ATPase (cHKA) during hyperaldosteronism suggests that it functions in both K+conservation and electrogenic Na+ absorption in the colon when Na+-conserving mechanisms are activated. To test this hypothesis, wild-type ( cHKA +/+) and cHKA-deficient ( cHKA −/−) mice were fed Na+-replete and Na+-restricted diets and their responses were analyzed. In both genotypes, Na+ restriction led to reduced plasma Na+ and increased serum aldosterone, and mRNAs for the epithelial Na+ channel (ENaC) β- and γ-subunits, channel-inducing factor, and cHKA were increased in distal colon. Relative to wild-type controls, cHKA −/− mice on a Na+-replete diet had elevated fecal K+ excretion. Dietary Na+restriction led to increased K+ excretion in knockout but not in wild-type mice. The amiloride-sensitive, ENaC-mediated short-circuit current in distal colon was significantly reduced in knockout mice maintained on either the Na+-replete or Na+-restricted diet. These results demonstrate that cHKA plays an important role in K+ conservation during dietary Na+ restriction and suggest that cHKA-mediated K+ recycling across the apical membrane is required for maximum electrogenic Na+ absorption.

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