OK Cell Line Research Articles

Serum sulfate level and Slc13a1 mRNA expression remain unaltered in a mouse model of moderate vitamin D deficiency.

Sulfate is essential for healthy foetal growth and neurodevelopment. The SLC13A1 sulfate transporter is primarily expressed in the kidney where it mediates sulfate reabsorption and maintains circulating sulfate levels. To meet foetal demands, maternal sulfate levels increase by twofold in pregnancy via upregulated SLC13A1 expression. Previous studies found hyposulfataemia and reduced renal Slc13a1 mRNA expression in rodent models with either severe vitamin D deficiency or perturbed vitamin D signalling. Here we investigated a mouse model of moderate vitamin D deficiency. However, serum sulfate level and renal Slc13a1 mRNA expression was not decreased by a moderate reduction in circulating vitamin D level. We confirmed that the mouse Slc13a1 5'-flanking region was upregulated by 1,25(OH)2D3 using luciferase assays in a cultured renal OK cell line. These results support the presence of a functional VDRE in the mouse Slc13a1 but suggests that moderate vitamin D deficiency does not impact on sulfate homeostasis. As sulfate biology is highly conserved between rodents and humans, we proposed that human SLC13A1 would be under similar transcriptional regulation by 1,25(OH)2D3. Using an online prediction tool we identified a putative VDRE in the SLC13A1 5'-flanking region but unlike the mouse Slc13a1 sequence, the human sequence did not confer a significant response to 1,25(OH)2D3 in vitro. Overall, this study suggests that moderate vitamin D deficiency may not alter sulfate homeostasis. This needs to be confirmed in humans, particularly during pregnancy when vitamin D and sulfate levels need to be maintained at high levels for healthy maternal and child outcomes.

The transcriptome of the Didelphis virginiana opossum kidney OK proximal tubule cell line.

The OK cell line derived from the kidney of a female opossum Didelphis virginiana has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, the genomic sequence for D. virginiana is not available and although a draft genome sequence for the opossum Monodelphis domestica (sequenced in 2012 by the Broad Institute) exists, transcripts sequenced from both species show significant divergence. The M. domestica sequence is not highly annotated, and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the D. virginiana OK cell line, we characterized its transcriptome via de novo transcriptome assembly and alignment to the M. domestica genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the M. domestica genome, were compared with publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT-specific ion transporters and other key proteins relevant for rodent and human PT function. Additionally, the sequence and expression data reported here provide an important resource for genetic manipulation and other studies on PT cell function using these cells.

Role of NaDC1 in Control of Urinary Excretion of α‐Ketoglutarate

Recent studies have suggested that urinary α‐Ketoglutarate (α‐KG) increases with alkali loads and decreases with acid loads. Also luminal α‐KG stimulates distal bicarbonate secretion via the Oxgr1 (GPR99) receptor.To investigate the role of sodium dependent dicarboxylate transporter, NaDC1, in regulation of α‐KG excretion, we studied NaDC1 knockout (KO) and Heterozygous (Het) mice on normal diet and with 72 hrs acid loading. Plasma and urine samples were analyzed by LC/Mass Spec.Also 14C‐α‐KG uptake was studied using HRPE cells overexpressing hNaDC1 (CUBS) and the proximal tubule (PT) OK cell line. OK cells express a novel calcium (Ca)‐sensitive dicarboxylate transport system.In mice, fractional excretion (FE) of α‐KG was dramatically increased in the KO and Het mice (WT 0.38+/‐0.05, Het 2.8+/‐1.3, KO 4.7+/‐0.9; p<0.05). Acidosis lowered FE significantly in all mice.In CUBS α‐KG uptake decreased when competed with the dicarboxylate succinate (Suc 2 mM) in both normal and <60 µM Ca buffers (0.64±0.11 to 0.25±0.10 and 0.63±0.15 to 0.29±0.11, p<0.01).In OK cells, α‐KG uptake increased when extracellular Ca was lowered to <60 µM (1.7±0.49 vs 2.5±0.6, p<0.01). Transport decreased when competed with Suc in both Ca buffers (1.3±0.39 vs 1.6±0.34, p<0.01).NaDC1 transports α‐KG and is the predominant, but not exclusive, mechanism of control of distal α‐KG delivery. Also α‐KG is transported via a Ca‐sensitive transporter in OK cells. Furthermore based on the FEs > 1.0, α‐KG is secreted into the PT simultaneously with reabsorption.Thus NaDC1 in the PT regulates acid‐base transport in the distal tubule via the control of α‐KG transport. NaDC1 represents a novel mechanism of PT–distal tubule communication in the kidney.NIH/NIDDK

NHERF2 Protein Mobility Rate Is Determined by a Unique C-terminal Domain That Is Also Necessary for Its Regulation of NHE3 Protein in OK Cells

Na(+)/H(+) exchanger regulatory factor (NHERF) proteins are a family of PSD-95/Discs-large/ZO-1 (PDZ)-scaffolding proteins, three of which (NHERFs 1-3) are localized to the brush border in kidney and intestinal epithelial cells. All NHERF proteins are involved in anchoring membrane proteins that contain PDZ recognition motifs to form multiprotein signaling complexes. In contrast to their predicted immobility, NHERF1, NHERF2, and NHERF3 were all shown by fluorescence recovery after photobleaching/confocal microscopy to be surprisingly mobile in the microvilli of the renal proximal tubule OK cell line. Their diffusion coefficients, although different among the three, were all of the same magnitude as that of the transmembrane proteins, suggesting they are all anchored in the microvilli but to different extents. NHERF3 moves faster than NHERF1, and NHERF2 moves the slowest. Several chimeras and mutants of NHERF1 and NHERF2 were made to determine which part of NHERF2 confers the slower mobility rate. Surprisingly, the slower mobility rate of NHERF2 was determined by a unique C-terminal domain, which includes a nonconserved region along with the ezrin, radixin, moesin (ERM) binding domain. Also, this C-terminal domain of NHERF2 determined its greater detergent insolubility and was necessary for the formation of larger multiprotein NHERF2 complexes. In addition, this NHERF2 domain was functionally significant in NHE3 regulation, being necessary for stimulation by lysophosphatidic acid of activity and increased mobility of NHE3, as well as necessary for inhibition of NHE3 activity by calcium ionophore 4-Br-A23187. Thus, multiple functions of NHERF2 require involvement of an additional domain in this protein.

NHE3 mobility in brush borders increases upon NHERF2-dependent stimulation by lyophosphatidic acid

The epithelial brush border (BB) Na(+)/H(+) exchanger NHE3 is associated with the actin cytoskeleton by binding both directly and indirectly to ezrin; indirect binding is via attachment to NHERF family proteins. NHE3 mobility in polarized epithelial cell BBs is restricted by the actin cytoskeleton and NHERF binding such that only approximately 30% of NHE3 in the apical domain of an OK cell line stably expressing NHERF2 is mobile, as judged by FRAP analysis. Given that levels of NHE3 are partially regulated by changes in trafficking, we investigated whether the cytoskeleton association of NHE3 was dynamic and changed as part of acute regulation to allow NHE3 trafficking. The agonist studied was lysophosphatidic acid (LPA), an inflammatory mediator, which acutely stimulates NHE3 activity by increasing the amount of NHE3 on the BBs by stimulated exocytosis. LPA acutely stimulated NHE3 activity in OK cells stably expressing NHERF2. Two conditions that totally prevented LPA stimulation of NHE3 activity only partially prevented stimulation of NHE3 mobility: the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the NHE3F1 double mutant which has minimal direct binding of NHE3 to ezrin. These results show that LPA stimulation of NHE3 mobility occurs in two parts: (1) PI3K-dependent exocytic trafficking to the BB and (2) an increase in surface mobility of NHE3 in BBs under basal conditions. Moreover, the LPA stimulatory effect on NHE3 mobility required NHERF2. Although NHE3 and NHERF2 co-precipitated under basal conditions, they failed to co-precipitate 30 minutes after addition of LPA, whereas the physical association was re-established by 50-60 minutes. This dynamic interaction between NHERF2 and NHE3 was confirmed by acceptor photobleaching Förster Resonance energy Transfer (FRET). The restricted mobility of NHE3 in BBs under basal conditions as a result of cytoskeleton association is therefore dynamic and is reversed as part of acute LPA stimulation of NHE3. We suggest that this acute but transient increase in NHE3 mobility induced by LPA occurs via two processes: addition of NHE3 to the BB by exocytosis, a process which precedes binding of NHE3 to the actin cytoskeleton via NHERF2-ezrin, and by release of NHERF2 from the NHE3 already localized in the apical membrane, enabling NHE3 to distribute throughout the microvilli. These fractions of NHE3 make up a newly identified pool of NHE3 called the 'transit pool'. Moreover, our results show that there are two aspects of LPA signaling involved in stimulation of NHE3 activity: PI3K-dependent stimulated NHE3 exocytosis and the newly described, PI3K-independent dissociation of microvillar NHE3 from NHERF2.

Regulation of Megalin Expression in Cultured Proximal Tubule Cells by Angiotensin II Type 1A Receptor- and Insulin-Mediated Signaling Cross Talk

Impairment of proximal tubular endocytosis of glomerular-filtered proteins including albumin results in the development of proteinuria/albuminuria in patients with chronic kidney disease. However, the mechanisms regulating the proximal tubular function are largely unknown. This study aimed to investigate the role of angiotensin II type 1A receptor (AT(1A)R)- and insulin-mediated signaling pathways in regulating the expression of megalin, a multiligand endocytic receptor in proximal tubule cells (PTCs). Opossum kidney PTC-derived OK cells that stably express rat AT(1A)R but are deficient in endogenous angiotensin II receptors (AT(1A)R-OK cells) were used for this study. Treatment of the cells with angiotensin II suppressed mRNA and protein expression of megalin at 3- and 24-h incubation time points, respectively. Cellular uptake and degradation of albumin and receptor-associated protein, megalin's endocytic ligands were suppressed 24 h after angiotensin II treatment. The AT(1A)R-mediated decrease in megalin expression was partially prevented by ERK inhibitors. Insulin competed with the AT(1A)R-mediated ERK activation and decrease in megalin expression. Inhibitors of phosphatidylinositol 3-kinase (PI3K), a major component of insulin signaling, also suppressed megalin expression, and activation of the insulin receptor substrate (IRS)/PI3K system was prevented by angiotensin II. Collectively the AT(1A)R-mediated ERK signaling is involved in suppressing megalin expression in the OK cell line, and insulin competes with this pathway. Conversely, the insulin-IRS/PI3K signaling, with which angiotensin II competes, tends to stimulate megalin expression. In conclusion, there is AT(1A)R- and insulin-mediated competitive signaling cross talk to regulate megalin expression in cultured PTCs.

Neutral amino acid transport mediated by ortholog of imino acid transporter SIT1/SLC6A20 in opossum kidney cells

Most neutral l-amino acid acids are transported actively across the luminal brush-border membrane of small intestine and kidney proximal tubule epithelial cells by a Na(+) cotransport system named B(0) that has been recently molecularly identified (B(0)AT1, SLC6A19). We show here that the opossum kidney-derived cell line OK also displays a Na(+)-dependent B(0)-type neutral l-amino acid transport, although with a slightly differing substrate selectivity. We tested the hypothesis that one of the two B(0)AT1-related transporters, SLC6A18 (ortholog of orphan transporter XT2) or SLC6A20 (ortholog of the recently identified mammalian imino acid transporter SIT1), mediates this transport. Anti-sense RNA to OK SIT1 (oSIT1) but not to OK XT2 (oXT2) inhibited Na(+)-dependent neutral amino acid transport induced by OK mRNA injected in Xenopus laevis oocytes. Furthermore, inhibition of oSIT1 gene expression in OK cells by transfection of siRNA and expression of shRNA selectively reduced the Na(+)-dependent uptake of neutral l-amino acids. Finally, expression of OK cell oSIT1 cRNA in X. laevis oocytes induced besides the transport of the l-imino acid l-Pro also that of neutral l-amino acids. Taken together, the data indicate that in OK cells SIT1 (SLC6A20) is not only an apical imino acid transporter but also plays a major role as Na(+)-dependent neutral l-amino acid transporter. A similar double role could be envisaged for SIT1 in mammalian kidney proximal tubule and small intestine.

Morphologic and functional alterations induced by low doses of mercuric chloride in the kidney OK cell line: ultrastructural evidence for an apoptotic mechanism of damage

Mercury produces acute renal failure in experimental animal models, but the mechanism of tubular injury has not completely been clarified. There is an increased interest in the role of apoptosis in the pathogenesis of renal diseases that result primarily from injury to renal tubular epithelial cells. However, detailed studies of morpho-functional alterations induced by mercuric chloride in kidney cell lines are scarce. This work characterizes these alterations in OK cell cultures. Morphological alterations were profiled using light microscopy, transmission electron microscopy, and confocal microscopy, as well as mitochondrial functional assays in the cells exposed to low concentrations of HgCl 2. At concentrations of 1 and 10 μM of HgCl 2 there were no morphological or ultrastructural alterations, but the mitochondrial function (MTT assay) and intracellular ATP content was increased, especially at longer incubation times (6 and 9 h). At 15 μM HgCl 2, both the mitochondrial activity and the endogenous ATP decreased significantly. At this concentration the OK cells rounded up, had increased number of cytoplasmic vacuoles, and detached from the cell monolayer. At 15 μM HgCl 2 ultrastructural changes were characterized by dispersion of the ribosomes, dilatation of the cisterns of the rough endoplasmic reticulum, increase of number of cytoplasmic vacuoles, chromatin condensation, invaginations of the nuclear envelope, presence of cytoplasmic inclusion bodies, and alterations in the size and morphology of mitochondria. At 15 μM HgCl 2 apoptotic signs included membrane blebbing, chromatin condensation, mitochondrial alterations, apoptotic bodies, and nuclear envelope rupture. Using confocal microscopy and the mitochondrial specific dye MitoTracker Red, it was possible to establish qualitative changes induced by mercury on the mitochondrial membrane potential after incubation of the cells for 6 and 9 h with 15 μM HgCl 2. This effect was not observed at short times (1 and 3 h) with this same concentration, neither with 1 and 10 μM HgCl 2 in all the studied times. Taken together, these findings indicate that low concentrations of HgCl 2 induce apoptosis by inhibiting mitochondrial function, and the OK cell line may be considered a useful tool for the study of programmed cell death involving mercurial species and other heavy metals.

Basolateral LAT-2 has a major role in the transepithelial flux of L-cystine in the renal proximal tubule cell line OK.

During renal reabsorption, the amino acid transporters b(o,+) and y(+)L have a major role in the apical uptake of cystine and dibasic amino acids and in the basolateral efflux of dibasic amino acids, respectively. In contrast, the transporters responsible for the basolateral efflux of the apically transported cystine are unknown. This study shows the expression of system L and y(+)L transport activities in the basolateral domain of the proximal tubule-derived cell line OK and the cloning of the corresponding LAT-2 and y(+)LAT-1 cDNAs. Stable transfection with a LAT-2 antisense sequence demonstrated the specific role of LAT-2 in the basolateral system L amino acid exchange activity in OK cells. This partial reduction of LAT-2 expression decreased apical-to-basolateral trans-epithelial flux of cystine and resulted in a twofold to threefold increase in the intracellular content of cysteine. In contrast, the content of serine, threonine, and alanine showed a tendency to decrease, whereas other LAT-2 substrates were not affected. This demonstrates that LAT-2 plays a major specific role in the net basolateral efflux of cysteine and points to LAT-2 as a candidate gene to modulate cystine reabsorption.

P-Aminohippurate transport at the apical membrane in the OK kidney epithelial cell line.

We investigated the characteristics of transport of an organic anion, p-aminohippurate (PAH), at the apical membrane in a kidney epithelial cell line OK. Efflux and uptake of [14C]PAH across the apical membrane were measured using OK cell monolayers grown on microporous membrane filters. PAH efflux to the apical side was greater than that to the basolateral side and significantly inhibited by probenecid. Diethyl pyrocarbonate (DEPC), an inhibitor of potential-sensitive organic anion transport, significantly decreased PAH efflux to the apical side. Moreover, PAH efflux to the apical side was significantly decreased on incubation with high potassium buffer, as compared with control condition. Extracellular pH and Cl- had no effect on PAH efflux across the apical membrane. PAH uptake from the apical side was inhibited by various organic anions, and the inhibition patterns of PAH uptake from the apical and basolateral sides by various dicarboxylates were similar. These results suggested that PAH efflux to the apical side in OK cells was mediated by a potential-sensitive transport system, but not by an anion exchanger. Moreover, PAH uptake from the apical side was mediated by a specific transport system, which interacts with various organic anions and dicarboxylates.

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.

Obligatory Amino Acid Exchange via Systems bo,+-like and y+L-like: A TERTIARY ACTIVE TRANSPORT MECHANISM FOR RENAL REABSORPTION OF CYSTINE AND DIBASIC AMINO ACIDS

Mutations in the rBAT gene cause type I cystinuria, a common inherited aminoaciduria of cystine and dibasic amino acids due to their defective renal and intestinal reabsorption (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). One important question that remains to be clarified is how the apparently non-concentrative system bo,+-like, associated with rBAT expression, participates in the active renal reabsorption of these amino acids. Several studies have demonstrated exchange of amino acids induced by rBAT in Xenopus oocytes. Here we offer evidence that system bo,+-like is an obligatory amino acid exchanger in oocytes and in the "renal proximal tubular" cell line OK. System bo, +-like showed a 1:1 stoichiometry of exchange, and the hetero-exchange dibasic (inward) with neutral (outward) amino acids were favored in oocytes. Obligatory exchange of amino acids via system bo,+-like fully explained the amino acid-induced current in rBAT-injected oocytes. Exchange via system bo,+-like is coupled enough to ensure a specific accumulation of substrates until the complete replacement of the internal oocyte substrates. Due to structural and functional analogies of the cell surface antigen 4F2hc to rBAT, we tested for amino acid exchange via system y+L-like. 4F2hc-injected oocytes accumulated substrates to a level higher than CAT1-injected oocytes (i.e. oocytes expressing system y+) and showed exchange of amino acids with the substrate specificity of system y+L and L-leucine-induced outward currents in the absence of extracellular sodium. In contrast to L-arginine, system y+L-like did not mediate measurable L-leucine efflux from the oocyte. We propose a role of systems bo,+-like and y+L-like in the renal reabsorption of cystine and dibasic amino acids that is based on their active tertiary transport mechanism and on the apical and basolateral localization of rBAT and 4F2hc, respectively, in the epithelial cells of the proximal tubule of the nephron.

Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells.

Escherichia coli isolates that cause meningitis in newborns are able to invade the circulation and subsequently cross the blood-brain barrier. One mechanism for traversing the blood-brain barrier might involve transcytosis through the endothelial cells. The ability of the meningitis isolate E. coli IHE3034, of serotype 018:K1:H7, to invade epithelial (T24) and endothelial (EA-hy926) cells was investigated by the standard gentamicin survival assay and by electron microscopy. Human bladder epithelial and endothelial cells were efficiently invaded by strain IHE3034, whereas epithelial human colon Caco-2 cells, canine kidney MDCK cells, and the opossum [correction of opposum] epithelial kidney cell line OK were not invaded. The ability to invade human epithelial cells of the bladder could also be demonstrated for several other newborn meningitis E. coli strains and one septicemic E. coli strain. Studies utilizing inhibitors which act on eukaryotic cells revealed a dependence on microfilaments as well as on microtubules in the process of E. coli IHE3034 entry into T24 and EA-hy926 cells. These results indicated that cell cytoskeletal rearrangements are involved in bacterial uptake and suggest that there are either two pathways (microtubule dependent and microfilament dependent) or one complex pathway involving both microtubules and microfilaments. The intracellular IHE3034 organisms were contained in a host-membrane-confined compartment mainly as single microorganisms. Intracellular replication of 1HE3034 was not detected, nor did the number of intracellular bacteria decrease significantly during a 48-h period. The ability of E. coli O18:K1 to invade and survive within certain eukaryotic cells may be another virulence factor of meningitis-associated E. coli.

The rBAT Gene Is Responsible for L-Cystine Uptake via the bo,+-like Amino Acid Transport System in a “Renal Proximal Tubular” Cell Line (OK Cells)

Several studies have shown that the cRNA of human, rabbit, or rat rBAT induces in Xenopus oocytes sodium-independent, high affinity uptake of L-cystine via a system b0,(+)-like amino acid exchanger. We have shown that mutations in rBAT cause type I cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-425; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). Apart from oocytes, no other expression system has been used for transfection of functional rBAT activity. Furthermore, the b0,(+)-like transport activity has not been clearly described in the kidney or intestine. Here, we report that a "proximal tubular-like" cell line derived from opossum kidney (OK cells) expresses an rBAT transcript. Poly(A)+ RNA from OK cells induced by system b0,(+)-like transport activity in oocytes. This was hybrid-depleted by human rBAT antisense oligonucleotides. A polymerase chain reaction-amplified cDNA fragment (approximately 700 base pairs) from OK cell RNA corresponds to an rBAT protein fragment 65-69% identical to those from human, rabbit and rat kidneys. We have also examined transport of l-cystine in OK cells and found characteristics very similar to the amino acid exchanger activity induced by rBAT cRNA in oocytes. Uptake of L-cystine was of high affinity, sodium-independent and shared with L-arginine and L-leucine. It was trans-stimulated by amino acids with the same specificity as rBAT-induced transport activity in oocytes. Furthermore, it was localized to the apical pole of confluent OK cells. To demonstrate that the rBAT protein is functionally related to this transport activity, we have transfected OK cells with human rBAT antisense and sense sequences. Transfection with rBAT antisense, but not with rBAT sense, resulted in the specific reduction of rBAT mRNA expression and b0,(+)-like transport activity. These results demonstrate that rBAT is functionally related to the L-cystine uptake via system b0,(+)-like in the apical pole of the renal OK cell line.

Bone-selective analogs of human PTH(1-34) increase bone formation in an ovariectomized rat model.

Intermittent parathyroid hormone (PTH) therapy increases bone mass. The purpose of this study was to determine if analogs of human PTH(1-34) (hPTH[1-34]), which differ from the native sequence in their receptor-activating properties, could promote bone formation in an ovariectomized (OVX) osteopenic rat model. We synthesized two hPTH(1-34) analogs with single substitutions for serine in the 3-position that in vitro are partial agonists in kidney. In the renal cell line OK, maximal cyclic adenosine monophosphate (cAMP) activation by [His(3)]hPTH(134) was 50%, and maximal cAMP activation by [Leu(3)]hPTH(1-34) was 20% of that produced by hPTH(1-34). Both analogs were full agonists in UMR-106 rat osteosarcoma cells and other bone-derived systems, but both had reduced potency compared with hPTh(1-34). Six-month-old retired breeder Sprague-Dawley rats were ovariectomized, and five animals underwent sham operation. On day 56 post-OVX, five sham-operated and five pre-PTH treatment OVX animals were sacrificed, and the remaining animals were randomized into 10 groups of six animals each. All other animals were injected with one of the hPTH analogs or hPTH(1-34) at 0, 4, 40, or 400 mu g/kg of body weight (BW)/day and were killed on day 84. Histomorphometry of the proximal tibia metaphysis and biochemical markers of bone turnover (osteocalcin and pyridinoline cross-links) were the primary endpoints. The cancellous bone volume was significantly lower at day 56 post-OVX (pretreatment) and at day 84 post-OVX (post-vehicle treatment) than at baseline. None of the compounds significantly increased the cancellous bone volume. Trabecular number declined after OVX and did not change with hPTH treatment. In contrast, the trabecular thickness declined after OVX but was higher after treatment with 40 mu g/kg of BW/day or 400 mu g/kg of BW/day of hPTH(1-34). In OVX rats, the mineralizing surface was higher than baseline at day 56 and fell toward control levels by day 84. All three peptides produced marked dose-related increases in the mineralizing surface and bone formation rates, but the two analogs were less potent than hPTH(1-34). Likewise, all peptides produced significant dose-related increases in the serum osteocalcin level. The osteoclast surface was not affected by OVX but was decreased with medium and high doses of hPTH(1-34). Pyridinoline cross-link excretion was not significantly affected by treatment with hPTH(1-34) but responded with a dose-dependent decrease to treatment with [His3]hPTH(1-34). These data suggest that bone selective analogs of hPTH(1-34) maintain the ability to induce bone formation but are less potent than hPTH(1-34).

H(+)-peptide cotransport in Madin-Darby canine kidney cells: expression and calmodulin-dependent regulation.

The transport of the dipeptide glycylsarcosine was studied in the kidney cell lines OK, LLC-PK1, and Madin-Darby canine kidney (MDCK), grown as confluent monolayers on impermeable plastic supports. Uptake of the dipeptide in OK and LLC-PK1 cells was slow, was not inhibited by other peptides, and was not influenced by an inwardly directed H+ gradient, indicating lack of expression of the H(+)-peptide cotransport system in these cells under our conditions. In contrast, uptake of the dipeptide in MDCK cells was rapid and was found to be stimulated by an inwardly directed H+ gradient. This stimulation was markedly reduced by the protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone. The H+ gradient-dependent uptake of glycylsarcosine was inhibited by dipeptides and tripeptides and by the beta-lactam antibiotic cephalexin but not by the amino acids glycine and leucine. The uptake was saturable and apparently occurred via a single transport system. The Michaelis-Menten constant for the system was 1.3 +/- 0.1 mM, and the maximal velocity was 13.3 +/- 0.7 nmol.30.min-1.mg protein-1. Treatment of MDCK cells with the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7), CGS-9343B, or calmidazolium inhibited the glycylsarcosine uptake by 40-50% in a time- and dose-dependent manner. In contrast, the uptake of alanine, leucine, glucose, and taurine was found to be stimulated by treatment with W-7. Kinetic analysis revealed that the inhibition of the peptide transport activity was mainly associated with a decrease of the maximal velocity of the system.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition by 5-HT of forskolin-induced cAMP Formation in the renal opossum Epithelial cell line OK: Mediation by a 5-HT 1B like receptor and antagonism by methiothepin

The functional activity of various 5-HT receptor agonists, including 5-CT, sumatriptan, CP 93, 129 and 1-naphtylpiperazine, and of drugs known to bind with high affinity to 5-HT 1B (pindolol, propranolol, cyanopindolol, SDZ 21,009 and isamoltane) or 5-HT 1D binding sites (yohimbine and rauwolscine) was measured at 5-HT receptors that are negatively coupled to adenylate cyclase in cultures of the renal epithelial cell line OK. 5-HT receptor-mediated inhibition of adenylate cyclase was studied by measuring inhibition of cAMP formation, induced by 100 μM forskolin. Besides 5-HT, various other compounds with affinity for 5-HT receptors behaved as agonists with the following rank order of potency: RU 24,969 > 5-CT > dihydroergotamine = 5-HT > CP 93,129 > d-LSD > 1-naphtylpiperazine > sumatriptan > TFMPP = mCPP > CGS 12066B = metergoline > methysergide. The β-adrenergic receptor blockers cyanopindolol, SDZ 21,009, (−)-pindolol and (−)-propranolol, and the α 2-adrenergic blockers yohimbine and rauwolscine yielded agonist activity at nanomolar and micromolar concentrations, respectively. Isamoltane acted as a partial agonist. Methiothepin was the only compound that antagonised the OK cell 5-HT receptor-mediated inhibition of forskolin-induced cAMP formation. We conclude that the OK cell 5-HT receptor has properties consistent with a 5-HT 1B receptor, although differences are apparent with regard to potencies of some compounds. Methiothepin is probably the only effective antagonist at 5-HT 1B receptor sites, whereas the described putative 5-HT 1B receptor antagonists have to be considered as partial agonists, yielding agonist or antagonist activity depending on the system that is studied.

Na(+)-dependent sulfate transport in opossum kidney cells is DIDS sensitive.

Sulfate transport was examined in OK/E cells, a clonal subline of opossum kidney cells that express several differentiated functions of the proximal tubule. Extracellular Na+ stimulated [35S]sulfate uptake five- to sixfold. Hill analysis demonstrating the dependence of sulfate uptake on Na+ concentration yielded a Hill coefficient of 1.5 and a Michaelis constant (KNa+) of 23 mM. Na(+)-dependent sulfate uptake was increased by lowering the pH from 7.4 to 6.4, decreased by raising the pH to 8.4 and inhibited by a 10-fold molar excess of SO3(2-), S2O3(2-) and CrO4(2-), but not by phosphate. The Na(+)-mediated component of sulfate uptake was saturable and kinetic parameters were estimated [Michaelis constant (Km) = 2.4 +/- 0.2 mM and maximum velocity (Vmax) = 125 +/- 15 pmol.mg protein-1.min-1]. Omitting extracellular Cl- resulted in a significant increase in the affinity of the carrier for sulfate (Km = 0.5 mm), without changing Vmax, consistent with competitive inhibition by Cl-. Na(+)-dependent sulfate uptake in OK/E cells was also inhibited by HCO3- [half-maximal inhibitory concentration (IC50) = 7 mM], 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, IC50 = 0.9 microM), 0.5 mM picrylsulfonic acid and 0.1 mM ethacrynic acid, but not by 1 mM amiloride. Na(+)-dependent, DIDS-sensitive sulfate uptake was also expressed in the parental OK cell line and was not influenced by serum or 3,3',5-triiodo-L-thyronine. We conclude that Na(+)-dependent sulfate uptake in OK/E cells observes many of the features of Na(+)-sulfate cotransport in the renal brush-border membrane and provides a useful model to investigate the regulation of renal sulfate transport.

The Use of the Epithelial Kidney OK Cell Line for Studying the Nephrotoxicity of Anticancer Platinum Coordination Complexes

Nephrotoxicity is one of the most important dose-limiting side-effects of cis-diaminedichloroplatinum (II) (cDDP) in humans. Quiescent OK cells grown in hormonally-defined, serum-free medium in the total absence of antibiotics were used to study the in vitro nephrotoxicity of three platinum complexes which produce different renal toxicity in vivo: cDDP, its stereoisomer trans-diaminedichloroplatinum (II) (tDDP), and cis-diamine-1,1-cyclobutane-dicarboxylateplatinUm (II) (CBDCA). The uptake and cytotoxicity of these compounds at concentrations of 3-l,600μM and their impact on DNA and protein synthesis, glucose uptake, Na+-K+-ATPase and succinate dehydrogenase activities, as well as intracellular total glutathione level, were measured. The results showed that the cytotoxicity ranking of these three compounds, assessed by the LDH release method, was not in agreement with their in vivo nephrotoxic potentials (tDDP > cDDP > CBDCA00 in OK cells versus cDDP > CBDCA > tDDP in vivo). cDDP and tDDP showed similar uptakes at all the concentrations studied, which demonstrated that their cytotoxic potential was not directly related to intracellular levels of platinum. At non-cytotoxic concentrations, both cDDP and CBDCA decreased DNA and protein synthesis and, to a lesser extent, Na+-K+-ATPase activity, whereas no effect on glucose uptake and succinate dehydrogenase activity (a mitochondrial marker enzyme) was observed. Nevertheless, 5–10 times greater concentrations of CBDCA were required to induce effects similar to those induced by cDDP. Our results did not show a rapid and early depletion of intracellular glutathione after exposure to cDDP or CBDCA. tDDP exhibited the characteristics of a non-specific cytotoxic chemical which was unable to markedly inhibit the biochemical and functional parameters studied at non-cytotoxic concentrations. These results underline the key role of the inhibition of synthetic activities in the pathogenesis of cDDP-induced and CBDCA-induced nephrotoxicity in OK cells.

Receptor-mediated endocytosis of albumin in cultured opossum kidney cells: a model for proximal tubular protein reabsorption.

The accumulation of fluorescein (FITC)-labelled bovine albumin was measured against the extracellular-fluid-phase marker FITC-inulin within confluent monolayers of the opossum kidney cell line OK. Fluorescence and electron microscopic pictures show that FITC-albumin is taken up by endocytosis and appears in a vesicular intracellular distribution. The uptake of FITC-albumin was quantified by measuring the cell-adherent fluorescence fluorimetrically. FITC-albumin uptake shows a time- and concentration-dependent saturation kinetics in contrast to the non-saturable FITC-inulin uptake, and exceeds the latter more than tenfold at low concentrations. Half-maximum saturation occurs at 20-30 mg/l. Initial FITC-albumin uptake/mg protein is stimulated by cell maturation, being six-to sevenfold higher in the confluent than in the subconfluent state, while FITC-inulin uptake is unchanged. Both an elevation of ambient osmolality to 600-750 mOsm/kg and disruption of the cytoskeleton by cytochalasin B (0.1 mmol/l) reduce initial FITC-albumin uptake by 50%-60% in a non-additive fashion. Albumin endocytosis is reduced both in acidic (pH 5.4) and alkaline (pH 8.4) medium, but does not depend on extracellular sodium, calcium or chloride. High concentrations of fetal calf serum or unlabelled albumin reduce FITC-albumin endocytosis dose-dependently. The present study is the first to investigate both the protein uptake and the fluid-phase endocytosis in a cultured proximal tubular cell line, using these cells as a model system-for proximal tubular protein reabsorption.