OK Cells Research Articles

Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane.

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.

Carrier-mediated uptake of cisplatin by the OK renal epithelial cell line

The purpose of this study was to investigate whether transport of cis-diamminedichloroplatinum II (cisplatin) across renal epithelial cell line OK cells is mediated by the organic cation transport system. OK cell monolayers cultured on permeable membranes were incubated with 100 μM cisplatin on the apical or basolateral side, and the cellular accumulation and the transport of cisplatin across the monolayer were measured. The accumulation from the basolateral medium and the basolateral-to-apical transport of cisplatin were higher than the accumulation from the apical medium and the apical-to-basolateral transport, respectively. The cell monolayers were incubated with different concentrations of cisplatin (0.02∼3 mM) in the basolateral medium. The relationship between the cisplatin concentrations in the medium and in the cells revealed that cisplatin accumulation tended to be saturable. The basolateral-to-apical transport of cisplatin was increased when the pH of the apical medium was decreased, with a concomitant decrease in the accumulation of cisplatin. Coincubation of cisplatin with tetraethylammonium (TEA), a typical substrate for the organic cation transporter, significantly decreased the accumulation and transport of cisplatin from the basolateral medium. These results suggest that the uptake and basolateral-to-apical transport of cisplatin are mediated by not only simple diffusion but also by the organic cation transport system.

Proximal tubular phosphate reabsorption: molecular mechanisms.

Renal proximal tubular reabsorption of P(i) is a key element in overall P(i) homeostasis, and it involves a secondary active P(i) transport mechanism. Among the molecularly identified sodium-phosphate (Na/P(i)) cotransport systems a brush-border membrane type IIa Na-P(i) cotransporter is the key player in proximal tubular P(i) reabsorption. Physiological and pathophysiological alterations in renal P(i) reabsorption are related to altered brush-border membrane expression/content of the type IIa Na-P(i) cotransporter. Complex membrane retrieval/insertion mechanisms are involved in modulating transporter content in the brush-border membrane. In a tissue culture model (OK cells) expressing intrinsically the type IIa Na-P(i) cotransporter, the cellular cascades involved in "physiological/pathophysiological" control of P(i) reabsorption have been explored. As this cell model offers a "proximal tubular" environment, it is useful for characterization (in heterologous expression studies) of the cellular/molecular requirements for transport regulation. Finally, the oocyte expression system has permitted a thorough characterization of the transport characteristics and of structure/function relationships. Thus the cloning of the type IIa Na-P(i )cotransporter (in 1993) provided the tools to study renal brush-border membrane Na-P(i) cotransport function/regulation at the cellular/molecular level as well as at the organ level and led to an understanding of cellular mechanisms involved in control of proximal tubular P(i) handling and, thus, of overall P(i) homeostasis.

MECHANISMS UNDERLYING RENAL TUBULAR ACIDOSIS AND ALBUMINURIA INDUCED BY CISPLATIN

The mechanisms underlying nephrotoxicity, specifically proximal tubular acidosis and albuminuria, induced by cisplatin (CDDP) were investigated. In the kidney, Na+/H+ exchanger and vacuolar (V)-H+-ATPase are two major H+ excretion systems located in the brush-border membrane. In brush-border membrane vesicles isolated from CDDP-treated rats, the activity of V-H+-ATPase, but not Na+/H+ exchanger, decreasedm arkedly. In OK cells, CDDP treatment induced intracellular acidification. Thus, the inhibition of V-H+-ATPase would be involved in the proximal tubular acidosis induced by CDDP. V-H+-ATPase, as well as chloride channel ClC-5, in the endosomal membrane is important for the endocytic system. In OK cells, endocytosis of albumin was inhibited by CDDP, as well as by inhibitors for V-H+-ATPase and ClC-5. In vivo, CDDP administration to rats induced albuminuria, with maximum effect observed at 2-3 days after administration. Such a pattern was comparable with the inhibition of CDDP on V-H+-ATPase. Similar pattern was also observed for the urinary excretion of vitamin D binding protein, which is specifically taken up by the proximal tubular cells via megalin-mediated endocytosis. Taken together, the inhibition of V-H+-ATPase by CDDP would result in not only proximal tubular acidosis but also albuminuria (proteinuria).

Kinetic analysis of p-aminohippurate transport in the OK kidney epithelial cell line.

Kinetic analysis of p-aminohippurate transport in the OK kidney epithelial cell line.

Cadmium Inhibits Albumin Endocytosis in Opossum Kidney Epithelial Cells

Chronic exposure to cadmium results in proteinuria. To gain insights into the mechanism by which cadmium inhibits the protein transport in the renal proximal tubule, we investigated the effects of cadmium on the receptor-mediated endocytosis of albumin, using fluorescein isothiocyanate-labeled bovine serum albumin (FITC-albumin) as a model substrate and opossum kidney cell line (OK cell) as a proximal tubular cell model. Cell monolayers grown to confluence were treated with 100 μM CdCl2 for 60 min at 37°C, washed, and tested for FITC-albumin uptake (37°C) and surface binding (4°C). The amounts of FITC-albumin uptake and binding were quantified by fluorimetrically determining the cell-adherent fluorescence. Both the binding and uptake of FITC-albumin by OK cells appeared to be saturable and inhibitable by unlabeled albumin in the medium, indicating that specific receptor sites were involved. The uptake of FITC-albumin was inhibited by agents that interfere with the formation of endocytotic vesicle (hypertonic mannitol), endosomal acidification (NH4Cl), and vesicular trafficking (cytochalasin D and nocodazole), confirming that the uptake occurred via the process of receptor-mediated endocytosis. In cells treated with cadmium, the specific FITC-albumin uptake was significantly attenuated, and this was due to a reduction in Vmax and a rise in Km. These changes in kinetic parameters were similar to those induced by NH4Cl. The binding of FITC-albumin to the apical surface of OK cells was inhibited by cadmium treatment, and this was attributed to a reduction in Bmax. The values of Kd and its pH dependency were not altered by cadmium treatment. The formation of endocytotic vesicles, as judged by fluid phase endocytosis of FITC-inulin, was not changed by cadmium treatment. These results indicate that the receptor-mediated endocytosis of albumin is impaired in cadmium-treated OK cells most likely due to a defect in endosomal acidification and the attendant fall in ligand-receptor dissociation, which impairs receptor recycling and the overall efficiency of endocytosis.

Lectin-mediated transport of nanoparticles across Caco-2 and OK cells

Recent experiments by a number of workers have suggested that it may be possible to use various targeting molecules, which bind to the intestinal epithelium, to promote the uptake and transport of nanoparticles from the intestine to the circulation. We have used commercial nanoparticles to examine the effect of size, density and inhibitors on uptake of lectin-coated nanoparticles by epithelial cells. The degree of uptake was most influenced by the density of lectin on the particle, with size and type of lectin being less important. Uptake could be inhibited by the presence of specific sugars or free lectin. These studies should provide a good basis for the design of targetable biodegradable drug-loadable particles suitable for oral delivery.

Regulation of the renal Na-HCO(3) cotransporter. XI. Signal transduction underlying CO(2) stimulation.

We have previously shown that CO(2) stimulation of the renal Na-HCO(3) cotransporter (NBC) activity is abrogated by general inhibitors of protein tyrosine kinases. The more selective inhibitor herbimycin also blocked this effect at concentrations known to preferentially inhibit Src family kinases (SFKs). We therefore examined a role for SFKs in CO(2)-stimulated NBC activity. To this end, we engineered OK cells to express the COOH-terminal Src kinase (Csk), a negative regulator of SFKs. CO(2) stimulated NBC activity normally in beta-galactosidase-expressing and untransfected control cells. In contrast, Csk-expressing cells had normal baseline NBC activity that was not stimulated by CO(2). CO(2) stimulation increased both total SFK activity and specific tyrosine phosphorylation of Src. The specific MEK1/2 inhibitor PD-98059 completely inhibited the CO(2) stimulation of NBC activity as well as the accompanying phosphorylation and activation of ERK1/2. Our data suggest the involvement of both SFKs, probably Src, and the "classic" MAPK pathway in mediating CO(2)-stimulated NBC activity in renal epithelial cells.

Neural cell line-specific regulatory DNA cassettes harboring the murine D1A dopamine receptor promoter.

Transcription in the human and rat D1A dopamine receptor genes proceeds from two distinct promoters in neuronal cells while only the downstream intronic promoter is active in renal cells. To investigate the utility of these promoters in the brain cell-specific expression of transgenes, we now studied the 5' flanking region of the murine D1A gene. We confirmed the presence of two functional promoters utilized for the tissue-specific regulation of this gene similar to its human and rat homologues. The cloned 1.4-kb genomic fragment spans nucleotides - 967 to + 384 relative to the first ATG codon and includes intron 1 between bases -534 to -420. Transient expression analyses using various chloramphenicol acetyltransferase constructs revealed that the murine D1A upstream promoter fused with the human D1A gene activator sequence ActAR1 has potent transcriptional activity in a D1A-expressing neuronal cell line but not in other cell lines tested including renal (OK cells), glial (C6) and hepatic (HepG2), suggesting that this hybrid construct harbors neural cell-specific elements. The availability of potent regulatory DNA cassettes harboring the murine D1A gene promoter could aid testing the neuronal-specific expression of transgenes in vivo.

Ochratoxin A-binding proteins in rat organs and plasma and in different cell lines of the kidney

In order to detect cellular proteins which bind the mycotoxin ochratoxin A (OTA) we coupled OTA covalently to horseradish peroxidase (HRP). The peroxidase activity of the conjugate was used to detect these proteins in Western (ligand) blot analysis. Only signals caused by OTA binding to proteins were viewable. HRP alone detected no proteins and OTA-HRP binding could be inhibited by free OTA. Several proteins from the rat intestine, liver, spleen, and kidney were detected by OTA. Also rat plasma proteins bind OTA which confirms previous findings. In all renal cell lines investigated (MDCK-C11, OK, LLC-PK1, IHKE, and SKPT) there are several proteins which bind OTA. Comparison of the PonceauS stain on the nitrocellulose sheet with the signal obtained from OTA-HRP unveiled proteins with high specific OTA binding. Especially, proteins with molecular masses between 55 and 60 kDa, 40 and 45 kDa and 25 and 30 kDa showed OTA binding in all samples. OTA was partially displaced by aspartame and phenylalanine from some but not all proteins. Binding to cytosolic and organellar proteins was comparable in all investigated cell lines. In the OK cell organellar compartment a 62 kDa protein is preferentially detected by OTA-HRP although virtually no protein band is detectable. In conclusion we have found a method to clearly detect proteins which bind OTA. With this new method we proved that OTA has the potential to bind to several proteins yet specific binding differs dramatically. Thus, highly specific binding of OTA possibly makes certain proteins a preferential target of OTA toxicity. Furthermore, binding contributes to intracellular accumulation of OTA, thus leading to a prolonged half life in the mammalian body and emphasises the toxic potential of this fungal metabolite.

Regulation of the Na/H Exchanger Regulatory Factor in OK Cells

The Na/H exchanger regulatory factor (NHE-RE), a recently cloned renal protein, is a necessary cofactor in protein kinase A-mediated inhibition of the renal brush border membrane Na/H exchanger. No studies to date, however, have examined the regulation of NHE-RF itself. The rabbit NHE-RF cDNA and an antibody to rabbit NHE-RF were used to study the effects of serum and cyclic adenosine monophosphate (cAMP) on the steady-state levels of NHE-RF mRNA and on the abundance and intracellular distribution of the protein in OK cells. Incubation of quiescent cells with serum was associated with a significant decrease in steady-state NHE-RF mRNA and protein abundance in the cytosolic and membrane fractions. Incubation of cells with cAMP for 6 h was associated with no change in NHE-mRNA at 24 h. There was, however, a 46% increase in protein abundance in the cytosolic fraction of the cell and a 43% decrease in the membrane fraction. Despite the decrease in membrane-associated NHE-RF in quiescent cells treated with serum of cAMP, there were no differences in either the basal rate of Na/H exchange transport or the inhibitory effect of the acute addition of cAMP on the transporter between experimental and control cells. These studies provide the first description of the regulation of NHE-RF. The results indicate that serum is associated with a decrease in NHE-RF mRNA and protein, while chronic exposure to cAMP is associated with an altered distribution of NHE-RF between the cytosolic and membrane fractions of OK cells.

Opioid agonists modify breast cancer cell proliferation by blocking cells to the G2/M phase of the cycle: involvement of cytoskeletal elements.

Opioids decrease cell proliferation in different systems including breast, prostate, lung, kidney, and intestine, through an interaction with opioid as well as other membrane-receptor systems (somatostatin, cholinergic), through an unidentified mechanism. Recently, we have reported an interaction of taxol with opioid membrane sites (BBRC 235, 201-204, 1997), and an involvement of opioids to the modification of actin cytoskeleton in renal OK cells (J Cell Biochem. [19981 70:60-69), indicating a possible action of the opioid effect. In the present work, we have examined the effect of two general opioid agonists (ethylketocyclazocine and etorphine) on the cell cycle, in human breast cancer T47D cells, as well as a possible modification of the cellular cytoskeleton under their action, in order to explain the antiproliferative effect of these agents. These two opioids produce a dose-dependent and reversible decrease of the proliferation of T47D cells, with a maximum attained at 10(-8) M. The addition of 10(-8) M of either opioid produced a significant increase of the number of cells arrested in the G2/M phase. Confocal laser microscopy revealed a modification of the actin and tubulin microfilaments, with a clear redistribution at the periphery of the cell, reversed by the addition of the general opioid antagonist diprenorphine. Furthermore, differences between the two opioids were obvious, attributed to the different receptor affinity of each agent. The observed redistribution of actin and tubulin cytoskeletal elements gives therefore a possible answer of the antiproliferative action of opioids. The modification of the cytoskeleton, directly involved to cell division, might provoke a "mechanical" obstacle, which could be the reason of the antiproliferative effect of these agonists. Furthermore, the observed tubulin-opioid interaction by opioids provides a possible explanation of the arrest at the G2/M phase of T47D cells under opioid treatment. Nevertheless, although the observed interaction of opioids with cytoskeletal elements gives a plausible answer of the antiproliferative effects of the agents, this might not be the only action of these agents in cell proliferation. Other, direct or indirect, genomic actions, which which remains to be elucidated, might be taken into consideration.

Lysophosphatidic acid-induced calcium mobilization and proliferation in kidney proximal tubular cells.

Patients with proteinuria tend to develop progressive renal disease with proximal tubular cell atrophy and interstitial scarring. It has been suggested that the nephrotoxicity of albuminuric states may be due to the protein molecule itself or by lipids, such as lysophosphatidic acid (LPA), that albumin carries. LPA was found to cause a transient increase in intracytoplasmic free Ca2+ ([Ca2+]i) in opossum kidney proximal tubule cells (OK) that was maximal at 100 microM LPA and was dose dependent with an EC50 of 2.6 x 10(-6) M. This Ca2+ mobilization was from both internal stores and across the plasma membrane and was pertussis toxin (PTX) insensitive. Treatment of OK cells with 100 microM LPA for 5 min was found to cause a twofold increase in [3H]thymidine incorporation and a three- to fivefold increase over control after 24 h. This was highly PTX sensitive and insensitive to pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A. These findings may be of significance in the progression of renal disease and indicate the potential importance of lipids in modulating proximal tubule cell function and growth.

A nucleoside-sensitive organic cation transporter in opossum kidney cells.

Renal secretion of organic cations and anions are pleiotropic, active processes in mammals. Some nucleosides such as deoxyadenosine (dAdo), 2-chlorodeoxyadenosine, and azidothymidine are secreted by human and rodent kidneys. Previous work (J. A. Nelson, J. F. Kuttesch, Jr., and B. H. Herbert. Biochemical Pharmacology 32: 2323-2327, 1983) indicated a role for the classic organic cation transporter (OCT) in the secretion of the dAdo analog, 2'-deoxytubercidin, by mouse kidney. Using [14C]tetraethylammonium bromide ([14C]TEA) as a substrate, we tested several renal cell lines for a nucleoside-sensitive OCT. American opossum kidney proximal tubule cells (OK) express a cimetidine-sensitive and metabolic-dependent ability to efflux TEA. Other classic OCT inhibitors and several nucleosides also inhibit TEA efflux by these cells in a manner reflecting structural specificity for the carrier. Inhibition of OCT by nucleosides is not a universal feature of OCTs, since TEA transport mediated by cloned rat kidney OCT2 in the Xenopus laevis oocyte system was not inhibited by the same nucleosides. In conclusion, OK cells appear to possess an OCT that may also transport some nucleosides by a novel carrier.

Type II Na-Pi cotransport is regulated transcriptionally by ambient bicarbonate/carbon dioxide tension in OK cells.

The purpose of the present study was to determine whether isohydric changes in HCO3 concentration and PCO2 directly affect apical Na-dependent Pi (Na-Pi) cotransport in OK cells (opossum kidney cell line). Cells were kept at either 44 mM NaHCO3/10% CO2, pH 7.4 (high-HCO3/CO2 condition), or 22 mM NaHCO3/5% CO2, pH 7.4 (low-HCO3/CO2 condition) (for 14-24 h). Incubation in lower HCO3/CO2 concentrations increased Na-Pi cotransport 1.5-fold. The increased Na-Pi cotransport was paralleled by a two- to threefold increased expression of the NaPi-4 transporter protein and a two- to threefold increase in NaPi-4 mRNA abundance. The increase in NaPi-4 mRNA could be completely prevented by incubation in the presence of a transcriptional inhibitor, suggesting that the increase in NaPi-4 mRNA results from an increased NaPi-4 mRNA transcription. In agreement, the NaPi-4 promoter activity was stimulated by 50% at lower HCO3/CO2 concentrations. In conclusion, our data demonstrate that isohydric changes in HCO3 concentration and PCO2 exert a significant, direct cellular effect on Na-Pi cotransport and NaPi-4 protein expression in OK cells by affecting NaPi-4 mRNA transcription.

The nephrotoxin ochratoxin A induces apoptosis in cultured human proximal tubule cells.

To test the apoptotic potential of the nephrotoxic mycotoxin ochratoxin A (OTA), we exposed human proximal tubule-derived cells (IHKE cells) for various times to OTA concentrations close to those occurring during dietary exposure (from 2 to 100 nmol/L) and investigated caspase 3 activation, chromatin condensation, and DNA fragmentation. OTA induced a time- and concentration-dependent activation of caspase 3: concentrations as low as 5 nmol/L OTA caused a slight but significant increase in caspase 3 activity after 7 days of OTA exposure. Exposure to 10 nmol/L OTA for 72 or 24 h led to a significantly increased activity of caspase 3 in human proximal tubule-derived cells. Radical scavengers such as N-acetylcysteine had no effect on OTA-induced caspase 3 activation. Chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethylester) (BAPTA-AM) also showed no effect. Exposure to 30 nmol/L or more OTA led to DNA fragmentation and chromatin condensation in IHKE cells. Cultured renal epithelial MDCK-C7 and MDCK-C11 or OK cells also showed increased caspase 3 activity after OTA exposure. We conclude that exposure to low OTA concentrations can lead to direct or indirect caspase 3 activation and subsequently to apoptosis in cultured human proximal tubule cells and in other renal epithelial cell lines of different origins.

Homologous regulation of the α2C‐adrenoceptor subtype in human hepatocarcinoma, HepG2

1. Previous studies of the regulation of the alpha2C-adrenoceptor in OK and in transfected cells have led to discrepant conclusions. In the present work, we examined the homologous regulation of the human alpha2C-adrenoceptor in the hepatocarcinoma cell-line, HepG2; a model which expresses this subtype spontaneously. 2. Short-period treatment of the cells with UK14304 provoked neither a diminution of the potency of the alpha2-agonist to inhibit forskolin-induced cyclic AMP-accumulation nor a change in the degree of receptor coupling to G-proteins. 3. Long-period exposure to UK14304 resulted in a large reduction of [3H]MK912 binding sites (55% decrease). The action of UK14304 was dose-dependent (EC50 = 190 +/- 45 nM), rapid (t1/2 = 4.2 h) and reversible. Receptor down-regulation was also observed with clonidine or (-)adrenaline (38 and 36% decrease, respectively) and was blocked by the addition of alpha2-antagonists. 4. Conversely to that observed with alpha2-agonists, treatment of the cells with RX821002 or yohimbine alone, but not with phentolamine, promoted a significant increase of the receptor expression. 5. The observed alterations of receptor density are not the reflection of changes at the alpha2C4 mRNA level. Estimation of the receptor protein turnover and measurement of its half-life demonstrated that down-regulation by alpha2-agonists and up-regulation by alpha2-antagonists, with inverse-agonist efficacy, are respectively the consequence of increased and decreased rate of receptor degradation. 6. In conclusion, our data show that alpha2C-adrenoceptor does not undergo desensitization but is down-regulated in HepG2. The lack of desensitization agrees with previous results obtained in cells transfected with the alpha2C4 gene, but not with observations made in OK cells. Inversely, down-regulation fits with results obtained in OK but not in transfected cells. The reasons for these discrepancies are discussed. Our results also demonstrated that certain alpha2-antagonists behave as inverse agonist on the HepG2 model and thus provide for the first time evidence of inverse efficacy of antagonists on a cellular model expressing physiological level of a wild-type alpha2-adrenoceptor.

Parathyroid hormone regulates the expression of rat osteoblast and osteosarcoma nuclear matrix proteins.

Parathyroid hormone (PTH) alters osteoblast morphology. How these changes in cell shape modify nuclear structure and ultimately gene expression is not known. Chronic exposure to rat PTH (1-34) [10 nM] attenuated the expression of 200, 190, and 160 kD proteins in the nuclear matrix-intermediate filament subfraction of the rat osteosarcoma cells, ROS 17/2.8 [Bidwell et al. (1994b): Endocrinology 134:1738-1744]. Here, we determined that these same PTH-responsive proteins were expressed in rat metaphyseal osteoblasts. We identified the 200 kD protein as a non-muscle myosin. Although the molecular weights, subcellular distribution, and half-lives of the 190 and 160 kD proteins were similar to topoisomerase II-alpha and -beta, nuclear matrix enzymes that mediate DNA topology, the 190 and 160 kD proteins did not interact with topoisomerase antibodies. Nevertheless, the expression of topoisomerase II-alpha, and NuMA, a component of the nuclear core filaments, was also regulated by PTH in the osteosarcoma cells. The 190 kD protein was selectively expressed in bone cells as it was not observed in OK opossum kidney cells, H4 hepatoma cells, or NIH3T3 cells. PTH attenuated mRNA expression of the PTH receptor in our cell preparations. These results demonstrate that PTH selectively alters the expression of osteoblast membrane, cytoskeletal, and nucleoskeletal proteins. Topoisomerase II-alpha, NuMA, and the 190 and 160 kD proteins may direct the nuclear PTH signalling pathways to the target genes and play a structural role in osteoblast gene expression.

Bidirectional transport of cadmium across apical membrane of renal epithelial cell lines via H +-antiporter and inorganic anion exchanger

The purpose of this study was to investigate whether cadmium (Cd) efflux across the apical membrane of renal epithelial cells is mediated via a H +-antiport system, and to confirm the re-uptake of Cd from the apical membrane via an inorganic anion exchanger. LLC-PK 1 and OK cell monolayers cultured on permeable membranes were incubated with 1 μM CdCl 2 added to the basolateral medium, and the transport of Cd from the basolateral to apical medium and the accumulation of Cd in the monolayers were measured. Cd transport was increased by lowering the pH of the apical medium and was accompanied by a decrease in Cd accumulation. Coincubation with N′-methylnicotinamide or cisplatin, which act as substrates of the H +-antiport systems, decreased Cd transport and increased Cd accumulation in a concentration-dependent manner. To confirm the re-uptake of Cd, LLC-PK 1 cell monolayers were pretreated with 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), a specific inhibitor of an inorganic anion exchanger, before incubation with CdCl 2. Pretreatment with DIDS significantly increased Cd transport and decreased Cd accumulation at an apical medium pH of 7.4, but not at pH 5.5. These results suggest bidirectional transport of Cd across the apical membrane of renal epithelial cells via a H +-antiport system (efflux) and an inorganic anion exchanger (influx), depending on the pH of the apical side.

CALCIUM OXALATE CRYSTAL ATTACHMENT TO CULTURED KIDNEY EPITHELIAL CELL LINES

Purpose Cultured kidney epithelial cell lines have frequently been used in urolithiasis research, and in particular in studies related to the interactions between stone crystals and cell membranes. There is evidence that when epithelial cell lines are transformed or serially passed to immortalize them, they experience changes in both cell physiology and morphology. Stone research utilizing cell cultures is frequently necessary due to the lack of an animal model for spontaneous stone disease. However, the interpretation of these cell culture research studies might be clouded by any significant differences in cell physiology between primary cells and continuous cell cultures. Therefore, the present study was conducted to compare calcium oxalate monohydrate (COM) crystal attachment to two primary kidney epithelial cell lines and to various continuous cell lines. Materials and Methods The cell lines surveyed were primary mouse proximal tubule cells (pMPT), primary inner medullary collecting duct cells (pIMCD), semi-continuous inner medullary collecting duct cells (cIMCD), BSC-1 cells, COS-1 cells, LLC-PK 1 cells, MDCK cells, NRK-52E cells, and OK cells. All cell lines were cultured under identical conditions and the amount of COM attachment was measured using radioactive labeled COM crystals. Results COM crystal interaction with continuous kidney epithelial cells varied by a factor of two among the different cell lines. In general, cells that grew as regular, confluent cell monolayers, such as pMPT, pIMCD and cIMCD cells, exhibited the lowest levels of crystal attachment. Neither changes in membrane fluidity nor loss of normal epithelial cell membrane asymmetry seemed to correlate well with crystal attachment. After nine days of continuous cell culture, COM attachment to cIMCD cells dropped by 61 percent while crystal attachment to MDCK cells remained unchanged. It is unclear what makes these cell lines more resistant to crystal attachment compared to continuous cell lines. Conclusions The significant difference in COM attachment between primary kidney epithelial cells and continuous epithelial cell cultures and the apparent differences in growth morphology between primary and continuous cell cultures must be considered when selecting a cell line for use in kidney stone research. Comparison of cIMCD cells and MDCK cells during extended culture time revealed one possible explanation for the differences in COM attachment: the formation of a mature, end-differentiated, non-dividing cell monolayer could protect the cells from crystal attachment.