Transcobalamin II expression is regulated by transcription factor(s) binding to a hexameric sequence (TGGTCC) in the promoter region of the gene.

Transcobalamin II (TCII) is a plasma protein that transports cobalamin to tissues for cellular uptake by receptor-mediated endocytosis. Human umbilical vein endothelial cells (HUVEC) in culture constitutively express TCII. However, in other cell lines, TCII expression is dependent on high cell density. ECV304, a cell line with some properties of HUVEC, expresses TCII only when seeded at high density. An electrophoretic mobility-shift assay using nuclear extract from such high-density-seeded ECV 304 cells shifted a 24-bp oligonucleotide probe to generate an unique slow moving band that was competed out by unlabeled probe. This unique band was not observed with nuclear extract from low-density-seeded ECV304 cells. A 3(') sequence, 5(')-TGGTCC-3('), in the 24-bp oligonucleotide was identified as the binding site for the nuclear protein(s) because this band was not competed out when the hexameric sequence was scrambled to 5(')-CTTCTT-3('). Binding of a transcription factor(s) to this hexamer, that is located 121bp upstream of the transcription start site, appears to be essential for the regulated or constitutive expression of TCII.

Transcobalamin II synthesized in the intestinal villi facilitates transfer of cobalamin to the portal blood.

This study was designed to identify the cellular component of the intestinal villus where transcobalamin II (TCII) is synthesized, because this protein provides an essential function in the intestinal absorption of vitamin B(12) (cobalamin, Cbl). When a segment of proximal or distal small intestine of the guinea pig is cultured in medium containing [(57)Co]Cbl, TCII-[(57)Co]Cbl appears within 15 min. Northern blot analysis of RNA from both proximal and distal small intestine identified the TCII transcript. In situ hybridization of the distal ileum with (35)S-labeled TCII antisense transcript localized grains predominantly in crypts and in the lower third and central core of the villi. Grains were also evident at the base of the enterocytes in close apposition with the vascular network, whereas few grains appeared in the apical region of the columnar cells. This study provides evidence that TCII is constitutively expressed in the intestinal villi where vascular endothelium is abundant. In the distal ileum, where the intrinsic factor (IF) receptor is expressed, after uptake of IF-Cbl and the subsequent binding of free Cbl to TCII synthesized in the villi, the TCII-Cbl complex enters the microcirculation and passes into the portal blood.

Effect of gentamicin on the lysosomal system of cultured human proximal tubular cells. Endocytotic activity, lysosomal pH and membrane fragility.

Gentamicin treatment results in significant changes in lysosomal morphology and enzyme activity in renal tubular epithelium both in vivo and in vitro. In this study, cultured human proximal tubular cells (PTC) were treated with gentamicin (0, 0.01, 0.1, and 1.0 mg/ml) for 3, 7, 10 and 14 days, and the endocytotic activity, pH, and membrane fragility of the lysosomal system were examined. Fluorescein isothiocyanate-labeled dextran (FITC-dextran) was used to estimate endocytotic activity and intralysosomal pH. The fragility of isolated lysosomes was estimated by the release of N-acetyl-beta-glucosaminidase (NAG, EC3.2.1.30) into the medium. Gentamicin content was measured and correlated with the changes seen in lysosomal function. Gentamicin treatment caused a slight decrease in the rate with which human PTC accumulated FITC-dextran and a slight increase in intralysosomal pH. Treatment of human PTC with NH4Cl, a lysosomotropic compound, significantly increased the lysosomal pH; the NH4Cl-induced increase in the lysosomal pH of gentamicin-treated PTC, however, was not significantly different from control (0 mg gentamicin/ml). Lysosomes isolated from human PTC cultures released NAG upon incubation for 60 min at 37 degrees. There was no significant effect on the fragility of lysosomes isolated from cultures exposed to gentamicin for less than or equal to 7 days. Significantly increased fragility was seen, however, after 10 days of treatment with 1.0 mg gentamicin/ml and especially after a 14-day exposure to 0.01, 0.1, and 1.0 mg gentamicin/ml. Human PTC accumulated 0.47, 2.05 and 10.30 micrograms gentamicin/mg protein with 10 days of exposure to 0.01, 0.1 and 1.0 mg gentamicin/ml medium respectively. Gentamicin treatment associated with increased numbers of morphologically altered lysosomes, i.e. myeloid bodies, did not affect significantly the endocytotic activity and pH of lysosomes in cultured human PTC. Prolonged exposure (14 days) of human PTC to gentamicin, however, did increase the fragility of lysosomes after isolation. The increased numbers of morphologically altered lysosomes with increased fragility were not associated with any significant in vitro cell death. Therefore, it would appear that these lysosomal alterations are not directly responsible for the in vivo nephrotoxicity.

Extracellular Ca2+-dependent elevation in cytosolic Ca2+ potentiates HgCl2-induced renal proximal tubular cell damage.

While normal fluctuations of cytosolic Ca2+ ([Ca2+]i) occur physiologically, the deregulation of cellular Ca2+ homeostasis leads to cellular injury. The contribution of [Ca2+]i to the process of cellular damage was assessed in a model system where HgCl2 was used to induce plasma membrane damage in renal tubular cells. In the presence of 1.37 mM extracellular Ca2+, HgCl2 (10-50 microM) induced a slow, dose-dependent, 4-6 fold increase in [Ca2+]i (as measured by Quin 2) by 10 min of exposure, which could be abolished by prior incubation of the cells with dithiothreitol. Correlates of cellular injury, i.e., decrease in cell viability, change in cellular morphology, such as bleb formation, membrane distortion and mitochondrial swelling, were induced after HgCl2 addition. The rate and dose-responses of these changes were similar to that of [Ca]i elevation. When cells were exposed to HgCl2 in the absence of added extracellular Ca2+, there was no increase in [Ca2+]i and both the rate and extent of cell damage were reduced. When Ca2+ was readded to the extracellular medium after HgCl2, there was a rapid elevation of [Ca2+]i, increased cell killing and bleb formation. The observed correlation between [Ca2+]i elevation, decreased cell viability and morphological aberrations in terms of (i) dose-dependency for HgCl2, (ii) requirement for high extracellular Ca2+, and (iii) rate of change, suggests that HgCl2-induced renal cell damage involves the entry of Ca2+ from the extracellular milieu which potentiates the progression of cellular injury.

Biochemical and morphological effects of gentamicin in human proximal tubular cells: effects on lipid and lipoprotein metabolism.

The effects of gentamicin, an antibiotic used extensively for antimicrobial therapy on the ultrastructure, binding, internalization, degradation, and cholesterol esterification of low-density lipoproteins, were investigated in cultured human proximal tubular cells. Cells were incubated with 0.3 mM gentamicin for 21 days with the following observations. Cells treated with gentamicin contained numerous "myeloid bodies." The binding, internalization, and degradation of 125I-labeled low-density lipoproteins ([125I]LDL) in cells treated with gentamicin was twofold lower than control cells. Pulse-chase experiments demonstrated that gentamicin did not impair the internalization of receptor-bound LDL and their subsequent transport to the lysosome. The relative amounts of [125I]LDL displaced by increasing concentrations of unlabeled LDL were the same in both gentamicin-treated and control cells. This pattern was reflected in the cell surface binding, internalization, and degradation of [125I]LDL. Gentamicin did not alter the degradation of [125I]LDL in cell homogenates at 4.0. The data suggest that gentamicin decreases the receptor-mediated endocytosis of LDL and subsequent lipid metabolism.

HgCl2-induced changes in cytosolic Ca2+ of cultured rabbit renal tubular cells.

Fura 2 was used to measure changes in cytosolic [Ca2+] ([Ca2+]i) in cultured rabbit kidney proximal tubule cells exposed to HgCl2. Treatment with 2.5-10 microM HgCl2 resulted in an extracellular [Ca2+] ([Ca2+]e)-independent 2- to 12-fold increase in [Ca2+]i above resting levels of about 100 nM. Treatment with 25-100 microM HgCl2 caused a rapid [Ca2+]e-independent 10- to 12-fold increase in [Ca2+]i within 1 min followed by a recovery to about 2-fold steady state by 3 min. With 25-100 microM HgCl2, both magnitude and rate of Ca2+ increase were similar, but recovery was greater with increasing doses. A slower, secondary increase in [Ca2+]i followed which varied with HgCl2 concentration and required [Ca2+]e. The first increase in [Ca2+]i represents release from intracellular pools. Calcium channel blockers, calmodulin inhibitors, and mitochondrial inhibitors do not alter the patterns of [Ca2+]i changes due to HgCl2. The recovery response with higher HgCl2 concentrations appears to be triggered by Hg2+ and not by the increased [Ca2+]i. Sulfhydryl modifiers N-ethylmaleimide, PCMB and PCMBS produced [Ca2+]e-independent [Ca2+]i increases similar to those induced by low HgCl2 concentrations. Cell killing with HgCl2 was about 50% greater with normal [Ca2+]e than with low [Ca2+]e, suggesting that [Ca2+]e influx is important in accelerating injury leading to cell death.

The effect of gentamicin on human renal proximal tubular cells.

Human renal proximal tubular cells were exposed to gentamicin (0, 0.1, 0.5, 1.0, 5.0, 10.0 mg/ml medium) for 3, 7, 10, and 14 days. Cells were counted and cell viability was estimated by lactate dehydrogenase release. In addition, brush border-associated (gamma-glutamyltransferase) and lysosomal (acid phosphatase, N-acetyl-beta-glucosaminidase, and sphingomyelinase) enzyme activities were measured (0.01, 1.0, 3.3 mg/ml gentamicin for 3, 7, 10 and 14 days). The number of cells did not change significantly after gentamicin treatment. Cell viability, however, significantly decreased after 3 and 7 days exposure to 5 and 10 mg/ml gentamicin. Total lactate dehydrogenase activity was significantly decreased at 7, 10, and 14 days exposure to gentamicin greater than or equal to 5.0 mg/ml. gamma-Glutamyltransferase, acid phosphatase, and sphingomyelinase were decreased at 3, 7, and 10 days exposure to gentamicin greater than or equal to 1.0 mg/ml. Continued exposure to gentamicin less than or equal to 1.0 mg/ml appeared to have little or no effect on the activity of these enzymes at 14 days. N-Acetyl-beta-glucosaminidase activity, in contrast, was elevated (120-140% control) in the gentamicin-treated (greater than or equal to 1.0 mg/ml) groups at all time periods studied. Thus, gentamicin exposure resulted in changes in some of the enzyme activities in human renal proximal tubular cell cultures, but longer exposure (14 days) to gentamicin (less than or equal to 1.0 mg/ml) resulted in a return to control levels of some activities.

Isolation, culture and characterization of human renal tubular cells.

Tubular cells have been isolated, characterized and cultured from more than 70 adult cadaver kidneys (postmortem time less than or equal to 12 hr.). Confluent monolayers were observed at 7 days after seeding (10(6) cells/ml.) and cells demonstrating normal human karyotypes have been passaged up to 6 times. Primary isolates and monolayer cultures were negative for Factor VIII activity, and strongly positive for gamma-glutamyltransferase activity and keratin. Ultrastructurally primary isolates consisted of cells with numerous mitochondria, microvilli, cytoplasmic filaments and well-developed endocytotic apparati. Monolayer cultures examined at 7, 14, 21 and 72 days demonstrated less prominent microvilli and the additional structures of desmosomes and cell junctions. Membrane-associated and cytosolic enzyme activities were measured up to 28 days in culture. The membrane-associated enzymes gamma-glutamyltransferase and alkaline phosphatase both exhibited approximately 10-fold decreases in activity during the 1st 7 days in culture. There was an approximately 5-fold increase in pyruvate kinase activity during the same time period, while fructose-1,6-bisphosphatase activity exhibited a 5-fold decrease. Glucose-6-phosphatase activity did not change during the 28 day culture period examined. From 7 to 28 days no further changes were noted in any of the enzyme activities measured. Decreased membrane-associated enzyme activity corresponded to the ultrastructural observation of less prominent microvilli. Increases in glycolytic enzyme activity and decreases in gluconeogenic enzyme activity may reflect the presence of glucose in the culture medium. The morphologic and biochemical evidence suggests that primary isolates and cultures are proximal tubule cells which should provide a well-defined in vitro human system for future studies.