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.

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.

The Na ؉ -HCO 3 ؊ 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 NH 2 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 COOHterminal 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.The Na ϩ -HCO 3 Ϫ cotransporter NBC1 mediated bicarbonate transport and is located on the basolateral membrane of the kidney proximal tubule and a number of other epithelia including pancreatic duct, gastric parietal cells, small intestine, and pulmonary alveolar cells (1-6). NBC1 functions in tandem with apical Na ϩ /H ϩ exchanger 3 and is essential for bicarbonate reabsorption in kidney proximal tubule (4,7,8). In pancreas, NBC1 is responsible for bicarbonate transport into pancreatic duct for eventual secretion into the pancreatic duct (4, 9, 10).Two well known variants of NBC1 are expressed in various epithelial tissues. These are the kidney (kNBC1) 1 and pancreatic (pNBC1) variants, which differ only in their NH 2 -terminal sequence, where amino acids 1-41 in kNBC1 have been replaced with 85 amino acids in pNBC1 (3). The carboxyl-terminal end of NBC1 is identical in kidney and pancreatic variants (1-3). Kidney NBC1 has a stoichiometry of three equivalents of bicarbonate for each sodium, whereas pancreatic NBC1 has a stoichiometry of two equivalents of bicarbonate per each sodium (11,12). This alteration in stoichiometry allows for the kidney NBC1 to function in the absorptive mode and for the pancreatic NBC1 to function in the secretory mode (4, 9 -12).In all of the tissues examined to date, NBC1 is found to be located on the basolateral membrane of epithelial cells (4 -6), strongly suggesting the presence of specific amino acid motif(s) that directs its targeting to the basolateral membrane. To identify possible motifs that ar...

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 (JHCO3 = 53.52 ± 4.61 pmol/min/mm in KO vs. 83.09 ± 9.73 in WT) and a ∼27 % reduction in volume reabsorption (Jv = 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.