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1.
Summary The aminoglycoside antibiotic streptomycin is a known nephrotoxin in vivo and a common component of cell culture media. The effects of streptomycin (100 μg/ml) on transepithelial electrical properties, glucose transport, glycolytic metabolism, and morphology were examined in primary proximal tubule cell cultures from winter flounder (Pseudopleuronectes americanus) kidney. Streptomycin treatment on either Days 2 to 12 or Days 8 to 13 abolished the transepithelial potential difference and short-circuit current across the monolayer but had no effect on transepithelial resistance in confluent 12 to 13-dcultures, suggesting the loss of active transepithelial transport. Consistent with these findings, mucosal-to-serosal glucose fluxes were greatly reduced in streptomycin-treated cultures and insensitive to the transport inhibitor phlorizin, indicating the absence of the apical Na-dependent glucose transport system associated with net glucose reabsorption. In addition to transport processes, antibiotic treatment also interfered with cellular energy metabolism as judged by the rapid reduction in glycolytic lactate production observed in the presence of streptomycin. Scanning and transmission electron microscopy revealed that streptomycin-treated culture were composed of cuboidal-to-columnar shaped cells which maintained intact tight junctions similar to control cultures. However, apical microvilli, the presumed sites of mucosal transport systems, were severely reduced in number in streptomycin-treated cultures. We concluded that streptomycin, at a dose commonly used in cell culture, inhibited the expression of differentiated function by flounder proximal tubule cell cultures. These cell cultures may provide a suitable model system for examination of the mechanisms of aminoglycoside nephrotoxicity. This investigation was supported by the University of Connecticut Research Foundation and by grant PCM-8003452 from the National Science Foundation, Washington, DC.  相似文献   

2.
Summary A primary rabbit kidney epithelial cell culture system has been developed which retains differentiated functions of the renal proximal tubule. In addition, the cells have a distinctive metabolism and spectrum of hormone responses. The primary cell were observed to retain in vitro a Na+-dependent sugar transport system (distinctive of the proximal segment of the nephron) and a Na+-dependent phosphate transport system. Both of these transport processes are localized on the apical membrane of proximal tubule cells in vivo. In addition, probenicid-sensitivep-aminohippurate (PAH) uptake was observed in basolateral membranes of the primary tubule cells, and the PAH uptake by these vesicles occurred at a rate that was very similar to that observed with membranes derived from the original tissue. Several other characteristics of the primary cells were examined, including hormone-sensitive cyclic AMP production and phosphoenolpyruvate carboxykinase (PEPCK) activity. Like the cells in vivo, the primary proximal tubule cells were observed to produce significant cyclic AMP in response to parathyroid hormone, but not in response to arginine vasopressin or salmon calcitonin. Significant PEPCK acivity was observed in the particulate fraction derived from a homogenate of primary rabbit kidney proximal tubule cells. This paper was presented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association held at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by Grant 9 RO1 DK40286-07 from the National Institutes of Health, Bethesda, MD, and NIH Research Career Development Award 1 K04 CA 0088-01 to M.T.  相似文献   

3.
Primary cultures of renal rabbit proximal tubule cells were initiated from a pure suspension of proximal tubule fragments. Proximal tubule cells were grown in a hormone-supplemented, serum-free medium containing low concentrations of antibiotics. Confluent monolayers exhibited multicellular dome formation, indicating the presence of transepithelial solute and water transport. Ultrastructural examination revealed a monolayer of polarized epithelial cells with tight junctions and sparse membraneous microvilli facing the culture medium. Time course biochemical characterization was performed using a palette of 12 enzymes, representative of important metabolic functions or pathways. Brush-border-associated enzymes (gamma-glutamyl transpeptidase and alanine aminopeptidase) were moderately reduced throughout the culture whereas alkaline phosphatase was markedly decreased at confluency. Mitochondrial and lysosomal marker enzymes were well preserved over the culture period. Glutathione-S-transferase activity remained stable during the 16-day culture period investigated. Glycolysis enzyme activities (lactate dehydrogenase and hexokinase) were enhanced, as a function of culture age. Na(+)-K(+)-ATPase activity rise was concomitant with the increase of glycolysis marker enzymes. In contrast, the gluconeogenesis marker enzyme, glucose-6-phosphatase, fell dramatically to reach a low level equivalent to 4% of the activity measured in isolated proximal tubules. Primary cultures exhibited several differentiated functions of the proximal tubule cell: (a) PTH alone was able to induce a significant stimulation of adenylate cyclase activity, unlike isoproterenol, thyrocalcitonin, and arginine vasopressin, and (b) sodium-dependent alpha-methylglucoside (AMG) transport was detected. This AMG uptake was selectively inhibited by phlorizin (5 X 10(-3) M), which is a competitive inhibitor of glucose uptake at the apical membrane. Complete characterization made it possible to investigate hitherto unexplored aspects of in vitro cultured proximal tubule cells. This primary culture model could provide a useful and reliable tool to investigate in vitro renal proximal tubule function, under normal conditions or after a drug-induced toxicity.  相似文献   

4.
Urate is a potent antioxidant at high concentrations but it has also been associated with a wide variety of health risks. Plasma urate concentration is determined by ingestion, production, and urinary excretion; however, factors that regulate urate excretion remain uncertain. The objective of this study was to determine whether cellular stress, which has been shown to affect other renal transport properties, modulates urate secretion in the avian renal proximal tubule. Chick kidney proximal tubule epithelial cell primary culture monolayers were used to study the transepithelial transport of radiolabeled urate. This model allowed examination of the processes, such as multidrug resistance protein 4 (Mrp4, Abcc4), which subserve urate secretion in a functional, intact, homologous system. Our results show that the recently implicated urate efflux transporter, breast cancer resistance protein (ABCG2), does not significantly contribute to urate secretion in this system. Exposure to a high concentration of zinc for 6 h induced a cellular stress response and a striking decrease in transepithelial urate secretion. Acute exposure to zinc had no effect on transepithelial urate secretion or isolated membrane vesicle urate transport, suggesting involvement of a cellular stress adaptation. Activation of AMP-activated protein kinase (AMPK), a candidate modulator of ATP-dependent urate efflux, by 5'-aminoimidazole-4-carboxamide 1-β-d-ribo-furanoside caused a decrease in urate secretion similar to that seen with zinc-induced cellular stress. This effect was prevented with the AMPK inhibitor compound C. Notably, the decrease in urate secretion seen with zinc-induced cellular stress was also prevented by compound C, implicating AMPK in regulation of renal uric acid excretion.  相似文献   

5.
Birds are uricotelic and, like humans, maintain high plasma urate concentrations (approximately 300 microM). The majority of their urate waste, as in humans, is eliminated by renal proximal tubular secretion; however, the mechanism of urate transport across the brush-border membrane of the intact proximal tubule epithelium during secretion is uncertain. The dominance of secretory urate transport in the bird provides a convenient model for examining this process. The present study shows that short hairpin RNA interference (shRNAi) effectively knocked down gene expression of multidrug resistance protein 4 (Mrp4; 25% of control) in primary monolayer cultures of isolated chicken proximal tubule epithelial cells (cPTCs). Control and Mrp4-shRNAi-treated cPTCs were mounted in Ussing chambers and unidirectional transepithelial fluxes of urate were measured. To detect nonspecific effects, transepithelial electrical resistance (TER) and sodium-dependent glucose transport (Iglu) were monitored throughout experiments. Knocking down Mrp4 expression resulted in a reduction of transepithelial urate secretion to 35% of control with no effects on TER or Iglu. Although electrical gradient-driven urate transport in isolated brush-border membrane vesicles was confirmed, potassium-induced depolarization of the plasma membrane in intact cPTCs failed to inhibit active transepithelial urate secretion. However, electrical gradient-dependent vesicular urate transport was inhibited by the MRP4 inhibitor MK-571 also known to inhibit active transepithelial urate transport by cPTCs. Based on these data, direct measure of active transepithelial urate secretion in functional avian proximal tubule epithelium indicates that Mrp4 is the dominant apical membrane exit pathway from cell to lumen.  相似文献   

6.
The mechanisms and control of transepithelial inorganic sulfate (Si) transport by primary cultures of chick renal proximal tubule monolayers in Ussing chambers were determined. The competitive anion, S2 O 3 2- (5 mM), reduced both unidirectional reabsorptive and secretory fluxes and net Si reabsorption with no effect on electrophysiological properties. The carbonic anhydrase (CA) inhibitor ethoxzolamide decreased net Si reabsorption approximately 45%. CAII protein and activity were detected in isolated chick proximal tubules by immunoblots and biochemical assay, respectively. Cortisol reduced net Si reabsorption up to approximately 50% in a concentration-dependent manner. Thyroid hormone increased net Si reabsorption threefold in 24 h, and parathyroid hormone (PTH) acutely stimulated net Si reabsorption approximately 45%. These data indicate that CA participates in avian proximal tubule active transepithelial Si reabsorption, which cortisol directly inhibits and T3 and PTH directly stimulate.  相似文献   

7.
In the intact kidney, renal proximal tubule cells accumulate p-aminohippurate (PAH) via a basolateral, probenecid- and sodium-sensitive transport system. Primary cultures of rabbit proximal tubule cells retain sodium-glucose co-transport in culture, but little is known about PAH transport in this system. Purified proximal tubule cells from a rabbit were grown in culture and assessed for PAH and alpha-methyl-D-glucoside uptake capacities as well as proximal tubule marker enzyme activities. Control PAH uptake on collagen-coated filters (20 +/- 3 pmol/mg protein.min; n = 8) was not significantly different from uptake in the presence of 1 mM probenecid (19 +/- 4 pmol/mg protein.min; n = 8). Uptake from the basal side of the cell was 3.9 +/- 0.7 times greater than that from the apical side. In multi-well plate studies, the uptake was significantly reduced by removing sodium from the medium and stimulated by coating the wells with collagen. Glutarate (10 mM) had no effect on the uptake of PAH. Other differentiated proximal tubule characteristics were retained in culture, including the ability to form domes and to transport glucose by a phlorizin-sensitive system. Phlorizin-sensitive 1 mM alpha-methyl-D-glucoside uptake was 134 +/- 42 pmol/mg protein.min (n = 7; P less than 0.02). The proximal tubule marker enzymes alkaline phosphatase and gamma-glutamyltranspeptidase, increased in activity in the cultures after confluence. It was concluded that whereas some differentiated properties were retained during primary culture of rabbit proximal tubule cells, the PAH transport system was selectively lost or modified from that present in the intact kidney.  相似文献   

8.
Summay Before the usefulness of a new in vitro model can be ascertained, the model must be properly defined and characterized. This study presents the growth rate and biochemical characteristics of rabbit renal proximal tubule cells in primary culture over a 2-wk culture period. When grown in a hormonally defined, antibiotic-free medium these cells form confluent monolayer cultures within 7 d after plating. Multicellular done formation, an indicator of transepithelial solute transport, was expressed after confluent cultures were formed. The activity of the cytosolic enzyme, lactate dehydrogenase, and the lysosomal enzyme,N-acetyl-glucosaminidase, increased 14- and 2-fold during the first 8 d of culture. respectively. In contrast, the activity of a brush border enzyme, alkaline phosphatase, decreased 85% within the first 8 d of culture. Release of these enzyme markers into the culture medium, which are routinely used to measure cytoxicity, stabilized after 8 d in culture. The ratio of cellular protein to DNA changed according to the state of cellular growth. Values rose from 0.035 mg protein/μg DNA in preconfluent cultures to 0.059 mg protein/μg DNA in confluent cultures. These results document the characteristics of a primary proximal tubule cell culture system for future studies in in vitro toxicology. This paper was resented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association held at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by grants GM 07145, The Johns Hopkins Center for Alternatives to Animal Testing, and a Sigma Xi Grants-in-Aid of Research Award.  相似文献   

9.
The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.  相似文献   

10.
Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.  相似文献   

11.
This study established two- and three-dimensional renal proximal tubular cell cultures of the endangered species bowhead whale (Balaena mysticetus), developed SV40-transfected cultures, and cloned the 61-amino acid open reading frame for the metallothionein protein, the primary binding site for heavy metal contamination in mammals. Microgravity research, modulations in mechanical culture conditions (modeled microgravity), and shear stress have spawned innovative approaches to understanding the dynamics of cellular interactions, gene expression, and differentiation in several cellular systems. These investigations have led to the creation of ex vivo tissue models capable of serving as physiological research analogs for three-dimensional cellular interactions. These models are enabling studies in immune function, tissue modeling for basic research, and neoplasia. Three-dimensional cellular models emulate aspects of in vivo cellular architecture and physiology and may facilitate environmental toxicological studies aimed at elucidating biological functions and responses at the cellular level. Marine mammals occupy a significant ecological niche (72% of the Earth's surface is water) in terms of the potential for information on bioaccumulation and transport of terrestrial and marine environmental toxins in high-order vertebrates. Few ex vivo models of marine mammal physiology exist in vitro to accomplish the aforementioned studies. Techniques developed in this investigation, based on previous tissue modeling successes, may serve to facilitate similar research in other marine mammals.  相似文献   

12.
Oxidation of [U14C]lactate to 14CO2 was measured in vitro, in nonperfused anatomically defined segments of rabbit proximal tubule (S1, proximal convoluted, and S2 and S3, proximal straight tubules). The rate of lactate oxidation was similar in S2 and S3 segments, and within the range of lactate oxidation rates measured in vivo. In contrast, the oxidation rate of S1 segments was significantly lower than that of S2 or S3. In proximal straight tubules, lactate oxidation was inhibited by incubation at 0 degrees C, or by application of 1 mM ouabain. To determine if the rate of transepithelial transport affected the rate of lactate oxidation, lactate oxidation was measured in proximal straight tubules after the lumen had been opened by perfusion with Ringer's containing 10 mM polyethylene glycol. No difference in lactate oxidation rate was observed between tubules with patent lumina and nonperfused tubules. These results suggest that the various segments of the renal proximal tubule have different metabolic characteristics, and that the rate of substrate oxidation is related to the activity of the Na+, K+-ATPase.  相似文献   

13.
14.
In primary cell cultures of the avian (Gallus gallus) renal proximal tubule parathyroid hormone and cAMP activation generate a Cl-dependent short circuit current (ISC) response, consistent with net transepithelial Cl secretion. In this study we investigated the expression and physiological function of the Na-K-2Cl (NKCC) transporter and CFTR chloride channel, both associated with Cl secretion in a variety of tissues, in these proximal tubule cells. Using both RT-PCR and immunoblotting approaches, we showed that NKCC and CFTR are expressed, both in proximal tubule primary cultures and in a proximal tubule fraction of non-cultured (native tissue) fragments. We also used electrophysiological methods to assess the functional contribution of NKCC and CFTR to forskolin-activated ISC responses in filter grown cultured monolayers. Bumetanide (10 μM), a specific blocker of NKCC, inhibited forskolin activated ISC by about 40%, suggesting that basolateral uptake of Cl is partially mediated by NKCC transport. In monolayers permeabilized on the basolateral side with nystatin, forskolin activated an apical Cl conductance, manifested as bidirectional diffusion currents in the presence of oppositely directed Cl gradients. Under these conditions the apical conductance appeared to show some bias towards apical-to-basolateral Cl current. Two selective CFTR blockers, CFTR Inhibitor 172 and GlyH-101 (both at 20 μM) inhibited the forskolin activated diffusion currents by 38-68%, with GlyH-101 having a greater effect. These data support the conclusion that avian renal proximal tubules utilize an apical CFTR Cl channel to mediate cAMP-activated Cl secretion.  相似文献   

15.
Summary Large numbers of kidney epithelial cells were cultured successfully from isolated dog proximal tubule segments. Cells in primary culture and in first passage retained the cystine-dibasic amino acid co-transporter system which is found in vivo and in freshly isolated proximal tubule segments. In contrast to other cultured cells, the cystine-glutamate anti-porter was absent in primary cultures. However, this anti-porter system seemed to be developing in cells in first passage. The intracellular ratio of cysteine:reduced glutathione (CSH:GSH) was maintained at 1∶36 in both primary cultures and in low passage cells. Incubation of cells in primary culture for 5 min at 37°C with 0.025 mM [35S]l-cystine resulted in incorporation of approximately 36 and 8.5% of the label into intracellular CSH and GSH, respectively. These cultured cells, therefore, seem to be an excellent model system for the eventual elucidation of a) the intricacies of cystine metabolism and b) regulation of 1) the cystine-dibasic amino acid co-transporter system and 2) the development of the cystine-glutamate anti-porter system. Supported by National Institutes of Health, Bethesda, MD, grant no. DK40555 and The National Kidney Foundation of the Delaware Valley.  相似文献   

16.
Summary The aim of this study was to better characterize rabbit proximal kidney tubule cells cultured on collagen IV-coated porous inserts, as compared to the same cells seeded in standard plastic wells. Total protein contents in confluent monolayers on permeable membranes were about twofold higher than those measured in confluent cultures in plastic wells. Microscopy examinations suggested that such a difference was probably due to a higher cell density and to an impressive development of the apical brush-border membrane. Moreover, measurement of unidirectional transport of p-aminohippuric acid and tetraethylammonium bromide confirmed the high polarization level of cultures on porous inserts. Results of methyl(α-d-[U-14C]glyco)pyranoside uptake suggested that cell phenotype was probably influenced by culture conditions. Analysis of different markers as a function of time in culture showed decreases of alkaline phosphatase (AP), γ-glutamyltranspeptidase (GGT), and Na+-K+-ATPase activities as well as increases in LDH, ATP, and glutathione levels, similar to those formerly reported for cells cultured in standard plastic plates. However, comparative data from 6-d-old monolayers have shown that AP, GGT, Na+-K+-ATPase, glutathione reductase (GRED), and selenium-dependent glutathione peroxidase (Se-GPX) activities were 2.8-, 2.6-, 1.6-, 1.2-, and 2.1-fold, respectively, better preserved on precoated permeable membranes. On the other hand, this paper reports for the first time in the literature that GRED and Se-GPX, two phase II detoxification enzymes, were well maintained in cultures of rabbit proximal kidney tubule cells. Our results show that culturing rabbit proximal kidney tubule cells on collagen IV-coated porous membranes was accompanied by an improvement of both morphological and biochemical properties of the cells.  相似文献   

17.
The Na(+) recirculation theory for solute-coupled fluid absorption is an expansion of the local osmosis concept introduced by Curran and analyzed by Diamond & Bossert. Based on studies on small intestine the theory assumes that the observed recirculation of Na(+) serves regulation of the osmolarity of the absorbate. Mathematical modeling reproducing bioelectric and hydrosmotic properties of small intestine and proximal tubule, respectively, predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1 (-/-) mice, and the inverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow. The model reproduces the volume responses of cells and lateral intercellular space (lis) following replacement of luminal NaCl by sucrose as well as the linear dependence of volume absorption on luminal NaCl concentration. Analysis of solvent drag on Na(+) in tight junctions provides explanation for the surprisingly high metabolic efficiency of Na(+) reabsorption. The model predicts and explains low metabolic efficiency in diluted external baths. Hyperosmolarity of lis is governed by the hydraulic permeability of the apical plasma membrane and tight junction with 6-7 mOsm in small intestine and < or = 1 mOsm in proximal tubule. Truly isosmotic transport demands a Na(+) recirculation of 50-70% in small intestine but might be barely measurable in proximal tubule. The model fails to reproduce a certain type of observations: The reduced volume absorption at transepithelial osmotic equilibrium in AQP1 knockout mice, and the stimulated water absorption by gallbladder in diluted external solutions. Thus, it indicates cellular regulation of apical Na(+) uptake, which is not included in the mathematical treatment.  相似文献   

18.
We have observed that, in renal proximal tubular cells, cardiotonic steroids such as ouabain in vitro signal through Na/K-ATPase, which results in inhibition of transepithelial (22)Na(+) transport by redistributing Na/K-ATPase and NHE3. In the present study, we investigate the role of Na/K-ATPase signaling in renal sodium excretion and blood pressure regulation in vivo. In Sprague-Dawley rats, high salt diet activated c-Src and induced redistribution of Na/K-ATPase and NHE3 in renal proximal tubules. In Dahl salt sensitive (S) and resistant (R) rats given high dietary salt, we found different effects on blood pressure but, more interestingly, different effects on renal salt handling. These differences could be explained by different signaling through the proximal tubular Na/K-ATPase. Specifically, in Dahl R rats, high salt diet significantly stimulated phosphorylation of c-Src and ERK1/2, reduced Na/K-ATPase activity and NHE3 activity, and caused redistribution of Na/K-ATPase and NHE3. In contrast, these adaptations were either much less effective or not seen in the Dahl S rats. We also studied the primary culture of renal proximal tubule isolated from Dahl S and R rats fed a low salt diet. In this system, ouabain induced Na/K-ATPase/c-Src signaling and redistribution of Na/K-ATPase and NHE3 in the Dahl R rats, but not in the Dahl S rats. Our data suggested that impairment of Na/K-ATPase signaling and consequent regulation of Na/K-ATPase and NHE3 in renal proximal tubule may contribute to salt-induced hypertension in the Dahl S rat.  相似文献   

19.
We have assessed the impact of increasing oxygen availability on cellular phenotype expression of rabbit proximal tubule cells in primary culture developed with variable glucose and/or insulin contents. To mitigate hypoxia at the cell/medium interface, cells were shaken for the whole culture duration and their expressed phenotype was compared with those expressed by static cultures. O2 and CO2 tensions were kept constant in the incubator atmosphere. Glycolysis and gluconeogenesis pathways, detoxication system, and mitochondrial, apical and basolateral membrane marker enzyme activities were assessed. This study showed that the induction of glycolysis which appear in primary cultures of proximal tubule cells may be partially prevented by continuously shaking the cultures. This effect was more marked in the presence of glucose, suggesting better substrate oxidation in shaken cultures.  相似文献   

20.
Bleich M  Shan QX 《生理学报》2007,59(4):443-453
K^+通道在上皮细胞内以极化的方式表达,形成一个庞大的膜蛋白家族。出于对主要依赖Na^+-K^+-ATPase而维持的细胞内跨膜K^+梯度的考虑,K^+通道在跨上皮细胞转运中的主要作用为:膜电位生成和K^+循环。本文以肾近端小管和胃壁上皮细胞转运为例简要阐述了K^+通道的作用。在这两个组织中,K^+通道活性限速跨上皮细胞转运,调节细胞体积。近年来,药理学工具和转基因动物的实验证实了对K^+通道的原先认知,并将研究深入到分子水平。K^+通道的分子结构挑战高亲和力药物分子的设计,及其多组织同时表达的两个典型特征阻碍了高活性、组织特异性小分子治疗的进展。然而,抑制K^+通道能阻断胃酸分泌等病理生理机制的深入研究,促进K^+通道药物用于胃病治疗和作为肾脏转运抑制剂用于肾脏相关疾病治疗。  相似文献   

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