Veedamali S. Subramanian

Disintegration of colonic epithelial tight junction in uremia: a likely cause of CKD-associated inflammation

BACKGROUND Inflammation is a constant feature and a major mediator of the progression of chronic kidney disease (CKD) and its numerous complications. There is increasing evidence pointing to the impairment of intestinal barrier function and its contribution to the prevailing inflammation in advanced CKD. Under normal condition, the intestinal epithelium and its apical tight junction prevent entry of the luminal microorganisms, harmful microbial by-products and other noxious contents in the hosts internal milieu. This study was designed to test the hypothesis that impaired intestinal barrier function in uremia must be due to disruption of the intestinal tight junction complex. METHODS Sprague-Dawley (SD) rats were randomized to undergo 5/6 nephrectomy (CKD) or sham-operation (control) and observed for 8 weeks. In a separate experiment, SD rats were rendered uremic by addition of 0.7% adenine to their food for 2 weeks and observed for an additional 2 weeks. Rats consuming a regular diet served as controls. The animals were then euthanized and their colons were removed and processed for expression of the key constituents of the tight junction complex using real-time polymerase chain reaction, western blot analysis and immunohistological examinations. RESULTS The CKD groups showed elevated plasma urea and creatinine, reduced creatinine clearance, thickened colonic wall and heavy infiltration of mononuclear leukocytes in the lamina propria. This was associated with marked reductions in protein expressions of claudin-1 (70-90%), occludin (50-70%) and ZO-1 (80-90%) in the colonic mucosa in both CKD models compared with the corresponding controls. The reduction in the abundance of the given proteins was confirmed by immunohistological examinations. In contrast, messenger RNA abundance of occludin, claudin-1 and ZO-1 was either unchanged or elevated pointing to the post-transcriptional/post-translational modification as a cause of the observed depletion of the tight junction proteins. CONCLUSION The study revealed, for the first time, that uremia results in depletion of the key protein constituents of the colonic tight junction, a phenomenon which can account for the impaired intestinal barrier function and contribute to the systemic inflammation in CKD.

Chronic alcohol consumption and intestinal thiamin absorption: effects on physiological and molecular parameters of the uptake process

Thiamin is essential for normal cellular functions, and its deficiency leads to a variety of clinical abnormalities. Humans and other mammals obtain the vitamin via intestinal absorption. The intestine is exposed to two sources of thiamin, a dietary and a bacterial (i.e., normal microflora of the large intestine) source. Chronic alcohol consumption is associated with thiamin deficiency, which is caused (in part) by inhibition in intestinal thiamin absorption. However, little is known about the physiological and molecular aspects of the intestinal thiamin uptake process that are affected by chronic alcohol use. To address these issues, we used rats fed an alcohol-liquid diet and human intestinal epithelial HuTu-80 cells chronically exposed to ethanol as model systems. The results showed that chronic alcohol feeding to rats led to a significant inhibition in carrier-mediated thiamin transport across both the jejunal brush-border membrane and basolateral membrane domains. This was associated with a significant reduction in level of expression of thiamin transporter-1 (THTR-1), but not THTR-2, at the protein and mRNA levels. Level of expression of the heterogenous nuclear RNA of THTR-1 in the intestine of alcohol-fed rats was also decreased compared with their pair-fed controls. Chronic alcohol feeding also caused a significant inhibition in carrier-mediated thiamin uptake in rat colon. Studies with HuTu-80 cells chronically exposed to ethanol also showed a significant inhibition in carrier-mediated thiamin uptake. This inhibition was associated with a reduction in level of expression of human THTR-1 and THTR-2 at the protein, mRNA, and transcriptional (promoter activity) levels. These studies demonstrate that chronic alcohol feeding inhibits intestinal thiamin absorption via inhibition of the individual membrane transport event across the polarized absorptive epithelial cells. Furthermore, the inhibition is, at least in part, mediated via transcriptional mechanism(s).

Folate uptake in the human intestine: promoter activity and effect of folate deficiency.

The intestinal folate absorption process occurs via a specialized mechanism that involves the reduced folate carrier (RFC). In humans, multiple variants of the hRFC (driven by multiple promoters) have been identified with variant I being the prominent form expressed in the intestine. While it is known that promoter B (pB) of hRFC drives the expression of this variant, little is known about the minimal region required for basal activity of this promoter in human intestinal epithelial cells. Also not known is whether folate absorption in the human intestine is up‐regulated during folate deficiency (as occur in animal studies), and if so, whether transcriptional mechanisms via activation of hRFC pB are involved in such regulation. To address these issues, we have used deletion constructs of the hRFC pB and determined their activity in two human intestinal epithelial cell lines: the colon‐derived Caco‐2 cells, and the duodenum‐derived HuTu‐80 cells. Our results showed that activity of hRFC pB to be significantly higher in Caco‐2 cells compared to HuTu‐80 cells, a finding that corresponds with a higher level of folate uptake and endogenous hRFC mRNA levels in the former compared to the latter cell type. The minimal region required for basal activity of hRFC pB in Caco‐2 cells was found to be encoded in a sequence between −1088 and −1043, while in HuTu‐80 cells it was encoded in a sequence between −1431 and −1088. Growing Caco‐2 cells in a folate deficient medium led to a significant and specific up‐regulation in folate uptake. This up‐regulation was associated with a parallel increase in hRFC protein and mRNA levels, and in the activity of hRFC pB. The most responsive sequence of pB to the effect of folate deficiency was found to be encoded in a sequence between −2016 and −1431, i.e., outside the minimal region of the pB. These results show that different minimal regions for hRFC pB are utilized by different intestinal epithelial cells. In addition, folate‐deficiency was found to up‐regulate folate uptake by human intestinal epithelial cells and that this regulation involves activation of hRFC pB. J. Cell. Physiol. 196: 403–408, 2003.

Cell Biology of the Human Thiamine Transporter-1 (hTHTR1) INTRACELLULAR TRAFFICKING AND MEMBRANE TARGETING MECHANISMS ,

The human thiamine transporter hTHTR1 is involved in the cellular accumulation of thiamine (vitamin B1) in many tissues. Thiamine deficiency disorders, such as thiamine-responsive megaloblastic anemia (TRMA), which is associated with specific mutations within hTHTR1, likely impairs the functionality and/or intracellular targeting of hTHTR1. Unfortunately, nothing is known about the mechanisms that control the intracellular trafficking or membrane targeting of hTHTR1. To identify molecular determinants involved in hTHTR1 targeting, we generated a series of hTHTR1 truncations fused with the green fluorescent protein and imaged the targeting and trafficking dynamics of each construct in living duodenal epithelial cells. Whereas the full-length fusion protein was functionally expressed at the plasma membrane, analysis of the truncated mutants demonstrated an essential role for both NH2-terminal sequence and the integrity of the backbone polypeptide for cell surface expression. Most notably, truncation of hTHTR1 within a region where several TRMA truncations are clustered resulted in intracellular retention of the mutant protein. Finally, confocal imaging of the dynamics of intracellular hTHTR1 vesicles revealed a critical role for microtubules, but not microfilaments, in hTHTR1 trafficking. Taken together, these results correlate hTHTR1 structure with cellular expression profile and reveal a critical dependence on hTHTR1 backbone integrity and microtubule-based trafficking processes for functional expression of hTHTR1.

Differential expression of human riboflavin transporters -1, -2, and -3 in polarized epithelia: a key role for hRFT-2 in intestinal riboflavin uptake.

Transport of riboflavin (RF) across both the brush border membrane (BBM) and basolateral membrane (BLM) of the polarized enterocyte occurs via specific carrier-mediated mechanisms. Although, three human riboflavin transporters (hRFTs), i.e., hRFT-1, hRFT-2 and hRFT-3 are expressed in the intestine, little is known about the cell surface domain(s) at which these specific hRFTs are expressed. Here, we used live cell confocal imaging of intestinal epithelial Caco-2 and renal MDCK cells to show that the hRFT-1 is mainly expressed at the BLM, hRFT-2 is exclusively expressed at the apical membrane, while hRFT-3 is mostly localized inside intracellular vesicular structures (with some expression at the BLM). Further the level of hRFT-2 mRNA expression in Caco-2 cells and in native human intestine is significantly higher than that of hRFT-1 and -3; hRFT-2 was also more efficient in transporting 3H-RF than hRFT-1 and -3. These findings implied an important role for hRFT-2 in intestinal RF uptake, a conclusion that was further supported by findings of hRFT-2 gene-specific siRNA knockdown investigation. These results show that members of the hRFT family are differentially expressed in polarized epithelia, and that the apically expressed hRFT-2 plays a key role in intestinal RF accumulation.

Intracellular Trafficking and Membrane Targeting Mechanisms of the Human Reduced Folate Carrier in Mammalian Epithelial Cells

The major pathway for cellular uptake of the water-soluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the reduced folate carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cytoplasmic NH2- or COOH-terminal domains of the protein. Further, the integrity of the hRFC backbone is critical for export of the polypeptide from the endoplasmic reticulum to the cell surface. This trafficking is critically dependent on intact microtubules because microtubule disruption inhibits motility of hRFC-containing vesicles as well as final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.

Mechanisms and regulation of vitamin C uptake : studies of the hSVCT systems in human liver epithelial cells

Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na(+) gradient, pH dependent, and saturable as a function of concentration over low and high micromolar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver, and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using short interfering RNA suggest that in HepG2 cells, decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a downregulation in ascorbic acid uptake not mediated by a single predicted PKC-specific amino acid phosphorylation site in hSVCT1 or hSVCT2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK, and Ca(2+)/calmodulin, but not by nitric oxide-dependent pathways. These studies are the first to determine the overall ascorbic acid uptake process and relative expression, regulation, and contribution of the hSVCT systems in human liver epithelial cells.

Polarized expression of members of the solute carrier SLC19A gene family of water-soluble multivitamin transporters: implications for physiological function

Humans lack biochemical pathways for the synthesis of the micro-nutrients thiamine and folate. Cellular requirements are met through membrane transport activity, which is mediated by proteins of the SLC19A gene family. By using live-cell confocal imaging methods to resolve the localization of all SLC19A family members, we show that the two human thiamine transporters are differentially targeted in polarized cells, establishing a vectorial transport system. Such polarization decreases functional redundancy between transporter isoforms and allows for independent regulation of thiamine import and export pathways in cells.

N-Glycosylation is required for Na+-dependent vitamin C transporter functionality

The human sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2) mediate cellular uptake of ascorbic acid. Both these transporters contain potential sites for N-glycosylation in their extracellular domains (Asn-138, Asn-144 [hSVCT1]; Asn-188, Asn-196 [hSVCT2]), however the role of N-glycosylation in transporter function is unexplored. On the basis of the result that tunicamycin decreased (14)C-ascorbic acid uptake in HepG2 cells, we systematically ablated all consensus N-glycosylation sites in hSVCT1 and hSVCT2 to resolve any effects on ascorbic acid uptake, transporter expression and targeting. We show that removal of individual N-glycosylation sites significantly impairs protein expression and consequently ascorbic acid uptake for hSVCT1 mutants (N138Q is retained intracellularly) and for hSVCT2 mutants (all of which reach the cell surface). N-Glycosylation is therefore essential for vitamin C transporter functionality.

Pancreatic beta cells and islets take up thiamin by a regulated carrier-mediated process: studies using mice and human pancreatic preparations

Thiamin is essential for the normal function of the endocrine pancreas, but very little is known about uptake mechanism(s) and regulation by beta cells. We addressed these issues using mouse-derived pancreatic beta-TC-6 cells, and freshly isolated primary mouse and human pancreatic islets. Results showed that thiamin uptake by beta-TC-6 cells involves a pH (but not Na+)-dependent carrier-mediated process that is saturable at both the nanomolar (apparent K(m) = 37.17 +/- 9.9 nM) and micromolar (apparent K(m) = 3.26 +/- 0.86 microM) ranges, cis-inhibited by thiamin structural analogs, and trans-stimulated by unlabeled thiamin. Involvement of carrier-mediated process was also confirmed in primary mouse and human pancreatic islets. Both THTR-1 and THTR-2 were found to be expressed in these mouse and human pancreatic preparations. Maintaining beta-TC-6 cells in the presence of a high level of thiamin led to a significant (P < 0.01) decrease in thiamin uptake, which was associated with a significant downregulation in level of expression of THTR-1 and THTR-2 at the protein and mRNA levels and a decrease in transcriptional (promoter) activity. Modulators of intracellular Ca2+/calmodulin- and protein-tyrosine kinase-mediated pathways also altered thiamin uptake. Finally, confocal imaging of live beta-TC-6 cells showed that clinical mutants of THTR-1 have mixed expression phenotypes and all led to impairment in thiamin uptake. These studies demonstrate for the first time that thiamin uptake by the endocrine pancreas is carrier mediated and is adaptively regulated by the prevailing vitamin level via transcriptional mechanisms. Furthermore, clinical mutants of THTR-1 impair thiamin uptake via different mechanisms.