Establishment and characterization of a cell line from the american opossum (Didelphys virginiana)

Koyama, Hideki; Goodpasture, Carll; Miller, Marcia M.; Teplitz, Raymond L.; Riggs, Arthur D.

doi:10.1007/bf02616032

Cited by 198 publications

(71 citation statements)

References 18 publications

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“…OK cells, an established proximal tubular epithelial cell line that is derived from the American opossum (23), retain many characteristics of proximal tubular cells and have been used extensively to investigate proximal tubular cell function (24) and the signaling effects of Cpeptide (11). OK cells that were obtained from Dr. J. Caverzasio (University Hospital, Geneva, Switzerland) were used in all experiments between passages 62 and 83.…”

Section: Cell Culturementioning

confidence: 99%

C-Peptide Signals via Gαi to Protect against TNF-α–Mediated Apoptosis of Opossum Kidney Proximal Tubular Cells

Al-Rasheed

Willars²,

Brunskill³

2006

Journal of the American Society of Nephrology

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Cell loss by apoptosis occurs in renal injury such as diabetic nephropathy. TNF-␣ is a cytokine that induces apoptosis and has been implicated in the pathogenesis of diabetic nephropathy. The aim was to investigate whether C-peptide or insulin could modulate TNF-␣-mediated cell death in opossum kidney proximal tubular cells and to examine the mechanism(s) of any effects observed. C-peptide and insulin protect against TNF-␣-induced proximal tubular cell toxicity and apoptosis. Cell viability was analyzed by methylthiazoletetrazolium assay; cell viability was reduced to 60.8 ؎ 2.7% of control after stimulation with 300 ng/ml TNF-␣. Compromised cell viability was reversed by pretreatment with 5 nM C-peptide or 100 nM insulin. TNF-␣-induced apoptosis was detected by DNA nick-end labeling and by measuring histone associated DNA fragments using ELISA. By ELISA assay, 300 ng/ml TNF-␣ increased apoptosis by 145.8 ؎ 4.9% compared with controls, whereas 5 nM C-peptide and 100 nM insulin reduced apoptosis to 81.6 ؎ 4.8 and 77.4 ؎ 3.1% of control, respectively. The protective effects of C-peptide and insulin were associated with activation of NF-B. Activation of NF-B by C-peptide was pertussis toxin sensitive and dependent on activation of G␣ i . Phosphatidylinositol 3-kinase but not extracellular signal regulated mitogen-activated protein kinase mediated C-peptide and insulin activation of NF-B. The cytoprotective effects of both C-peptide and insulin were related to increased expression of TNF receptor-associated factor 2, the product of an NF-B-dependent survival gene. These data suggest that C-peptide and/or insulin activation of NF-B-regulated survival genes protects against TNF-␣-induced renal tubular injury in diabetes. The data further support the concept of C-peptide as a peptide hormone in its own right and suggest a potential therapeutic role for C-peptide.

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Section: Cell Culturementioning

confidence: 99%

C-Peptide Signals via Gαi to Protect against TNF-α–Mediated Apoptosis of Opossum Kidney Proximal Tubular Cells

Al-Rasheed

Willars²,

Brunskill³

2006

Journal of the American Society of Nephrology

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“…OK cell line (25) clone 3B/2, between passages 5 and 30, was subcloned from the parental line for its better adaptability to a low phosphate medium and inhibition of phosphate transport by parathyroid hormone. Cells were grown in Dulbecco modified Eagle medium/ Nutrition Mix F-12 medium supplemented with 10% fetal calf serum, as described (18).…”

Section: Cell Culturementioning

confidence: 99%

Basolateral LAT-2 Has a Major Role in the Transepithelial Flux of L-Cystine in the Renal Proximal Tubule Cell Line OK

Fernández¹,

Torrents²,

Chillarón³

et al. 2003

Journal of the American Society of Nephrology

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Abstract. During renal reabsorption, the amino acid transporters b o,ϩ and y ϩ 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 ϩ 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 ϩ 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.In the kidney, most glomerulus-filtered amino acids are reabsorbed in the early proximal tubule (1). The amino acids are taken up through the brush border membrane and exit the epithelial cells through the basolateral membrane to the interstitial space. Two heteromeric amino acid transport exchangers (systems b o,ϩ and y ϩ L) have a major role in the reabsorption of cystine and dibasic amino acids (2-4). First, the heterodimer formed by rBAT and b o,ϩ AT is the amino acid transporter b o,ϩ , which mediates high-affinity uptake of cystine and dibasic amino acids coupled with the efflux of neutral amino acids (5-8) at the apical membrane of epithelial cells of the proximal tubule (9). Indeed, mutations in the rBAT (SLC3A1) gene cause type I cystinuria, and mutations in the b o,ϩ AT (SLC7A9) gene cause mainly non-type I and also type I cystinuria (recessive inherited aminoacidurias of cystine and dibasic amino acids) (6,10,11). Second, y ϩ LAT-1 dimerizes with 4F2hc to form the amino acid transporter y ϩ L, which mediates the efflux of cationic amino acids coupled with the influx of neutral amino acids plus sodium (12-14). Mutations in y ϩ LAT-1 (SLC7A7) cause lysinuric protein intolerance (15,16), an inherited aminoaciduria due to defective dibasic amino acid efflux from the basolateral membrane of proximal tubule epithelial cells (17). Thus, systems b o,ϩ and y ϩ L explain the trans-epithelial transport of dibasic amino acids. In contrast, the amino acid transporters that play a major role in the basolateral efflux of the apically taken-up cystine remain to be identified.Polarized OK cells, a proximal tubule-derived cell line from the American opossum, has been extensively used as a model for transport studies in renal epithelial cells. Indeed, OK cells express rBAT-associated system b o,ϩ transport activity in the...

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“…Our primary aim was to determine whether C-peptide has the capacity to signal in PTC, focusing especially on pathways involved in cell growth and the potential development of diabetic nephropathy. We used opossum kidney (OK) cells, an immortalised kidney PTC cell line [30] that retains many characteristics of the proximal tubule [31]. Many typical proximal tubular functions such as parathyroid hormone responses and endocytic uptake of proteins have been investigated using this cell type [32,33].…”

Section: Introductionmentioning

confidence: 99%

Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells

et al. 2004

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Aims/hypothesis. Proinsulin C-peptide is generally believed to be inert without any appreciable biological functions. However, it has been shown to modulate a variety of cellular processes important in the pathophysiology of diabetic complications. We therefore investigated the ability of C-peptide to stimulate intracellular signalling pathways in kidney proximal tubular cells, the altered activation of which may possibly be related to the development of diabetic nephropathy. Methods. Extracellular signal-regulated kinase (ERK) and Akt phosphorylation were evaluated by western blotting. ERK activity was measured by in vitro kinase assay. Intracellular Ca 2+ was evaluated by confocal imaging. The membrane and cytosol-associated fractions of protein kinase C (PKC) isoforms were evaluated by western blotting. Proliferation was assessed by thymidine incorporation assay. Results. Using the opossum proximal tubular kidney cell line as a model, we demonstrated that at high picomolar to low nanomolar concentrations, C-peptide stimulates extracellular signal-regulated mitogen-activated kinase (3.3±0.1-fold over basal at 3 minutes) and phosphatidylinositol 3-kinase (4.1±0.05-fold over basal at 5 minutes). ERK activation was attenuated by pre-treatment with a PKC inhibitor and abolished by pertussis toxin. Elevations of intracellular [Ca 2+ ] are seen in response to 5 nmol/l C-peptide with consequent activation of PKC-α. Pre-treatment with pertussis toxin abolished PKC-α. C-peptide is also a functional mitogen in this cell type, stimulating significantly increased cell proliferation. Proliferation was attenuated by wortmannin and pertussis toxin pretreatments. None of these effects is reproduced by scrambled C-peptide. Conclusions/interpretation. This study provides evidence that C-peptide, within physiological concentration ranges, stimulates many signalling pathways in opossum kidney cells.

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Establishment and characterization of a cell line from the american opossum (Didelphys virginiana)

Cited by 198 publications

References 18 publications

C-Peptide Signals via Gαi to Protect against TNF-α–Mediated Apoptosis of Opossum Kidney Proximal Tubular Cells

C-Peptide Signals via Gαi to Protect against TNF-α–Mediated Apoptosis of Opossum Kidney Proximal Tubular Cells

Basolateral LAT-2 Has a Major Role in the Transepithelial Flux of L-Cystine in the Renal Proximal Tubule Cell Line OK

Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells

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