Proximal tubule specific knockout of the Na⁺/H⁺ exchanger NHE3: effects on bicarbonate absorption and ammonium excretion.

The existing NHE3 knockout mouse has significant intestinal electrolyte absorption defects, making this model unsuitable for the examination of the role of proximal tubule NHE3 in pathophysiologic states in vivo. To overcome this problem, we generated proximal convoluted tubule-specific KO mice (NHE3-PT KO) by generating and crossing NHE3 floxed mice with the sodium-glucose transporter 2 Cre transgenic mice. The NHE3-PT KO mice have >80 % ablation of NHE3 as determined by immunofluorescence microscopy, western blot, and northern analyses, and show mild metabolic acidosis (serum bicarbonate of 21.2 mEq/l in KO vs. 23.7 mEq/l in WT, p < 0.05). In vitro microperfusion studies in the isolated proximal convoluted tubules demonstrated a ∼36 % reduction in bicarbonate reabsorption (J HCO3 = 53.52 ± 4.61 pmol/min/mm in KO vs. 83.09 ± 9.73 in WT) and a ∼27 % reduction in volume reabsorption (J v = 0.67 ± 0.07 nl/min/mm in KO vs. 0.92 ± 0.06 nl/min/mm in WT) in mutant mice. The NHE3-PT KO mice tolerated NH4Cl acid load well (added to the drinking water) and showed NH4 excretion rates comparable to WT mice at 2 and 5 days after NH4Cl loading without disproportionate metabolic acidosis after 5 days of acid load. Our results suggest that the Na(+)/H(+) exchanger NHE3 plays an important role in fluid and bicarbonate reabsorption in the proximal convoluted tubule but does not play an important role in NH4 excretion.

The role of aspartic acid residues 405 and 416 of the kidney isotype of sodium-bicarbonate cotransporter 1 in its targeting to the plasma membrane.

The NH(2) terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH(2)-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH(2) terminus of NBCe1A are important in its accurate targeting to the plasma membrane.

Impact of silver nanoparticles on human cells: effect of particle size.

This work investigated the cytotoxicities of three silver nanoparticles (SNPs) SNP-5, SNP-20 and SNP-50 with different sizes ( approximately 5 nm, approximately 20 nm and approximately 50 nm) using four human cell models (A549, SGC-7901, HepG2 and MCF-7). Endpoints included cell morphology, cell viability, cellular membrane integrity, oxidative stress and cell cycle progression. Observable deleterious effects on the cell morphologies and membrane integrity were induced by SNP-5 and SNP-20. SNPs elevated the ROS levels in cells and arrested the cells at S phase. Apoptosis occurred for 4-9% of the exposed cells. All these cellular responses as well as EC50 values were found to be size-dependent for the tested SNPs. Ultrastructural observations confirmed the presence of SNPs inside cells. Elemental analysis of silver in cells by ICP-MS showed that smaller nanoparticles enter cells more easily than larger ones, which may be the cause of higher toxic effects. The findings may assist in the design of SNP applications and provide insights into their toxicity.

Sequence- or position-specific mutations in the carboxyl-terminal FL motif of the kidney sodium bicarbonate cotransporter (NBC1) disrupt its basolateral targeting and alpha-helical structure.

The sodium-bicarbonate cotransporter NBC1 is targeted exclusively at the basolateral membrane. Mutagenesis of a dihydrophobic FL motif (residues 1013-1014) in the C-terminal domain disrupts the targeting of NBC1. In the present study, we determined the precise constraints of the FL motif required for basolateral targeting of NBC1 by expressing epitope-tagged wild-type and mutant NBC1 in MDCK cells and RNA-injected Xenopus oocytes and examining their subcellular localization. We assayed the functional activity of the mutants by measuring bicarbonate-induced currents in oocytes. Wild-type NBC1 (containing PFLS) was expressed exclusively on the basolateral membrane in MDCK cells. Reversal of the FL motif (PLFS) had no effect on basolateral targeting or activity. Shifting the FL motif one residue upstream (FLPS) resulted in mistargeting of the apical membrane but the FLPS mutant retained its functional activity in oocytes. Shifting the FL motif one residue downstream resulted in a mutant (PSFL) that did not efficiently translocate to the plasma membrane and was instead colocalized with the ER marker, protein disulfide isomerase (PDI). Analysis of circular dichroism (CD) revealed that a short peptide, 20 amino acid residues, of wild-type NBC1 contained a significant alpha-helical structure, whereas peptides in which the FL motif was reversed or C-terminally shifted were disordered. We therefore propose that the specific orientation and the precise location of the FL motif in the primary sequence of NBC1 are strict requirements for the alpha-helical structure of the C-terminal cytoplasmic domain and for targeting of NBC1 to the basolateral membrane.

Primary pulmonary adenocarcinoma with intestinal differentiation mimicking metastatic colorectal carcinoma: case report and review of literature.

Pulmonary adenocarcinoma with intestinal differentiation is rare and typically expresses proteins common to lung primaries. We report a case in a 51-year-old woman with a solitary 3.3-cm mass in the left lower lobe. Additional clinical investigation, including positron emission tomography scan with fluorine 18-labeled fluorodeoxyglucose, colonoscopy, and capsule endoscopy of her small bowel, revealed no evidence of tumor elsewhere. She underwent left lower lobectomy with mediastinal lymphadenectomy. Histologic examination revealed tall columnar cells without goblet cell differentiation arranged in a cribriform and acinar pattern with extensive central necrosis. Metastatic carcinoma was present in multiple hilar lymph nodes. Mediastinal lymph nodes were negative. Immunohistochemical stains demonstrated diffuse positivity for cytokeratin (CK) 20 and CDX-2 in neoplastic cells with negative staining for CK7 and thyroid transcription factor-1. CK7 expression has been documented in all 14 cases previously reported. This is the first description of pulmonary adenocarcinoma with intestinal differentiation with histopathologic and immunophenotypic findings indistinguishable from metastatic colorectal adenocarcinoma.

cAMP-dependent and cholinergic regulation of the electrogenic intestinal/pancreatic Na+/HCO3- cotransporter pNBC1 in human embryonic kidney (HEK293) cells.

BACKGROUND: The renal (kNBC1) and intestinal (pNBC1) electrogenic Na+/HCO3- cotransporter variants differ in their primary structure, transport direction, and response to secretagogues. Previous studies have suggested that regulatory differences between the two subtypes can be partially explained by unique consensus phosphorylation sites included in the pNBC1, but not the kNBC1 sequence. After having shown activation of NBC by carbachol and forskolin in murine colon, we now investigated these pathways in HEK293 cells transiently expressing a GFP-tagged pNBC1 construct. RESULTS: Na+- and HCO3-dependent pHi recovery from an acid load (measured with BCECF) was enhanced by 5-fold in GFP-positive cells compared to the control cells in the presence of CO2/HCO3-. Forskolin (10(-5) M) had no effect in untransfected cells, but inhibited the pHi recovery in cells expressing pNBC1 by 62%. After preincubation with carbachol (10(-4) M), the pHi recovery was enhanced to the same degree both in transfected and untransfected cells, indicating activation of endogenous alkalizing ion transporters. Acid-activated Na+/HCO3- cotransport via pNBC1 expressed in renal cells is thus inhibited by cAMP and not affected by cholinergic stimulation, as opposed to the findings in native intestinal tissue. CONCLUSION: Regulation of pNBC1 by secretagogues appears to be not solely dependent on its primary structure, but also on properties of the cell type in which it is expressed.

Deep juvenile xanthogranuloma presenting as a chest wall mass: a case report.

BACKGROUND: Juvenile xanthogranulotna (JXG) is a non-Langerhans cell histocytic proliferation that may appear as an extracutaneous deep-seated lesion and give a broad clinical dijffrrential diagnosis. We report the fine needle aspiration cytologv (FNAC) findings of deep JXG. CASE: A 5-month-old African-American boy was incidentally found to have a chest wall mass on a chest radiograph obtained for an unrelated medical problem. Subsequent computed tomographic scans documented a 3.8-cm soft tissue mass that involved the right chest wall centered around the fifth rib. A broad clinical differential diagnosis prompted FNA to evaluate the lesion. Aspirate smears of the mass exhibited numerous finely vacuolated histocytes, eosinophils, multinucleated giant cells and scattered Touton giant cells. Many of the histiocytes had reniform or grooved nuclei, resembling Langerhans cells. The histiocytes were immunoreactive for CD68 but were nonreactive for CD1a and S-100 protein. Subsequent excisional biopsy confirmed the diagnosis of JXG. In addition, the tumor was strongly immunoreactive for factor XIIIa. CONCLUSION: JXG should be considered in the diferential diagnosis of any histocytic/fibrohistiocytic soft tissue lesion of childhood, and this entity can be accurately diagnosed by FNAC and immunohistochemical findings.

Identification of a novel signal in the cytoplasmic tail of the Na+:HCO3- cotransporter NBC1 that mediates basolateral targeting.

The Na(+):HCO(3)(-) cotransporter NBC1 (SLC4A4, variant A, kidney specific) is located exclusively on the basolateral membrane of epithelial cells, implying that this molecule has acquired specific signals for targeting to the basolateral membrane. A motif with the sequence QQPFLS (positions 1010-1015) in the cytoplasmic tail of NBC1 was recently demonstrated to mediate targeting of NBC1 to the basolateral membrane. Here, we demonstrate that mutating the amino acid F (phenylalanine) or L (leucine) at positions 1013 or 1014 to alanine, respectively, resulted in the retargeting of NBC1 to the apical membrane. Furthermore, mutation of the FL motif to FF showed similar properties as the wild-type; however, mutation of the FL motif to LL showed significant intracellular retention of NBC1. Mutating the amino acids Q-Q-P and S (positions 1010-1011-1012 and 1015) to A-A-A and A, respectively, did not affect the membrane targeting of NBC1. Functional studies in oocytes with microelectrode demonstrated that the apically targeted mutants, as well as basolaterally targeted mutants, are all functional. We propose that the FL motif in the COOH-terminal tail of NBC1 is essential for the targeting of NBC1 to the basolateral membrane but is distinct from the membrane-targeting di-leucine motif identified in other membrane proteins.

Chloride/bicarbonate exchanger SLC26A7 is localized in endosomes in medullary collecting duct cells and is targeted to the basolateral membrane in hypertonicity and potassium depletion.

SLC26A7 is a Cl(-)/HCO(3)(-) exchanger that is expressed on the basolateral membrane and in the cytoplasm of two distinct acid-secreting epithelial cells: The A-intercalated cells in the kidney outer medullary collecting duct and the gastric parietal cells. The intracellular localization of SLC26A7 suggests the possibility of trafficking between cell membrane and intracellular compartments. For testing this hypothesis, full-length human SLC26A7 cDNA was fused with green fluorescence protein and transiently expressed in MDCK epithelial cells. In monolayer cells in isotonic medium, SLC26A7 showed punctate distribution throughout the cytoplasm. However, in medium that was made hypertonic for 16 h, SLC26A7 was detected predominantly in the plasma membrane. The presence of mitogen-activated protein kinase inhibitors blocked the trafficking of SLC26A7 to the plasma membrane. Double-labeling studies demonstrated the localization of SLC26A7 to the transferrin receptor-positive endosomes. A chimera that was composed of the amino terminal fragment of SLC26A7 and the carboxyl terminal fragment of SLC26A1, and a C-terminal-truncated SLC26A7 were retained in the cytoplasm in hypertonicity. In separate studies, SLC26A7 showed predominant localization in plasma membrane in potassium-depleted isotonic medium (0.5 or 2 mEq/L KCl) versus cytoplasmic distribution in normal potassium isotonic medium (4 mEq/L). It is concluded that SLC26A7 is present in endosomes, and its targeting to the basolateral membrane is increased in hypertonicity and potassium depletion. The trafficking to the cell surface suggests novel functional upregulation of SLC26A7 in states that are associated with hypokalemia or increased medullary tonicity. Additional studies are needed to ascertain the role of SLC26A7 in enhanced bicarbonate absorption in outer medullary collecting duct in hypokalemia and in acid-base regulation in conditions that are associated with increased medullary tonicity.

Missense mutations in Na+:HCO3- cotransporter NBC1 show abnormal trafficking in polarized kidney cells: a basis of proximal renal tubular acidosis.

The kidney Na(+):HCO(3)(-) cotransporter NBC1 is located exclusively on the basolateral membrane of kidney proximal tubule cells and is responsible for the reabsorption of majority of filtered bicarbonate. Two well-described missense mutations in NBC1, R510H and S427L, are associated with renal tubular acidosis (RTA). However, the exact relationship between these mutations and NBC1 dysregulation remains largely unknown. To address this question, cDNAs for wild-type kidney NBC1 and its mutants R510H and S427L were generated, fused in frame with NH(2) terminally tagged GFP, and transiently expressed in Madin-Darby canine kidney cells. In parallel studies, oocytes were injected with the wild-type and mutant NBC1 cRNAs and studied for membrane expression and activity. In monolayer cells grown to polarity, the wild-type GFP-NBC1 was exclusively localized on the basolateral membrane domain. However, GFP-NBC1 mutant R510H was detected predominantly in the cytoplasm. GFP-NBC1 mutant S427L, on the other hand, was detected predominantly on the apical membrane with residual cytoplasmic retention and basolateral membrane labeling. In oocytes injected with the wild-type or mutant GFP-NBC1 cRNAs, Western blot analysis showed that wild-type NBC1 is predominantly localized in the membrane fraction, whereas NBC1-R510H mutant was predominantly expressed in the cytoplasm. NBC1-S427L mutant was mostly expressed in the membrane fraction. Functional analysis of NBC1 activity in oocytes by membrane potential recording demonstrated that compared with wild-type GFP-NBC1, the GFP-NBC1 mutants H510R and S427L exhibited significant reduction in activity. These findings suggest that the permanent isolated proximal RTA in patients with H510R or S427L mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly retained in the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.

Identification of a carboxyl-terminal motif essential for the targeting of Na+-HCO-3 cotransporter NBC1 to the basolateral membrane.

The Na+-HCO3- cotransporter NBC1 is located exclusively on the basolateral membrane and mediates vectorial transport of bicarbonate in a number of epithelia, including kidney and pancreas. To identify the motifs that direct the targeting of kidney NBC1 to basolateral membrane, wild type and various carboxyl-terminally truncated kidney NBC1 mutants were generated, fused translationally in-frame to GFP, and transiently expressed in kidney epithelial cells. GFP was linked to the NH2 terminus of NBC1, and labeling was examined by confocal microscopy. Full-length (1035 aa) and mutants with the deletion of 3 or 20 amino acids from the COOH-terminal end of NBC1 (lengths 1032 and 1015 aa, respectively) showed strong and exclusive targeting on the basolateral membrane. However, the deletion of 26 amino acid residues from the COOH-terminal end (length 1010 aa) resulted in retargeting of NBC1 to the apical membrane. Expression studies in oocytes demonstrated that the NBC1 mutant with the deletion of 26 amino acid residues from the COOH-terminal end is functional. Additionally, the deletion of the last 23 amino acids or mutation in the conserved residue Phe at position 1013 on the COOH-terminal end demonstrated retargeting to the apical membrane. We propose that a carboxyl-terminal motif with the sequence QQPFLS, which spans amino acid residues 1010-1015, and specifically the amino acid residue Phe (position 1013) are essential for the exclusive targeting of NBC1 to the basolateral membrane.