Adaptation of Opossum Kidney Cells to Luminal Phosphate: Effects of Phosphonoformic Acid and Kinase Inhibitors

Thomas, Linto; Wagner, Carsten A.; Biber, Jürg; Hernando, Nati

doi:10.1159/000443432

Cited by 8 publications

(11 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our data in Npt2a-I-treated OK cells demonstrate, compared with vehicle, a significantly higher K m with no significant change in the apparent V max , consistent with a competitive pattern of inhibition. OK cells respond to increasing concentrations of P i in the media with inverse changes on Na ϩ -dependent P i uptake and Npt2a expression (47,58). Regarding the former, our data confirm these findings.…”

Section: F548supporting

confidence: 85%

“…The medium was changed every 3 days, and cells were split 1:3 once per week by trypsinization. Low-Pi (0.1 mmol/L) or high-Pi (2 mmol/L) medium was made using Pi-free medium (GIBCO) (47,58) supplemented with the desired Pi concentrations (Na2HPO4/NaH2PO4, pH 7.4) along with penicillin-streptomycin (50 IU/mL) and HEPES (20 mmol/L, GIBCO). Experiments were done between passages 45 and 60.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

PF-06869206 is a selective inhibitor of renal P_itransport: evidence from in vitro and in vivo studies

Thomas

Xue

Tomilin

et al. 2020

American Journal of Physiology-Renal Physiology

Self Cite

View full text Add to dashboard Cite

Plasma phosphate (Pi) levels are tightly controlled, and elevated plasma Pi levels are associated with an increased risk of cardiovascular complications and death. Two renal transport proteins mediate the majority of Pi reabsorption, the Na+-phosphate cotransporters Npt2a and Npt2c, with Npt2a accounting for 70-80% of Pi reabsorption. The aim of this study was to determine the in vitro effects of a novel Npt2a inhibitor (Npt2a-I, PF-06869206) in opossum kidney (OK) cells as well as determine its selectivity in vivo in Npt2a knockout (Npt2a-/-) mice. In OK cells, Npt2a-I caused dose-dependent reductions of Na+-dependent Pi uptake (IC50: ~1.4 μmol/L), while the unselective Npt2 inhibitor phosphonoformic acid (PFA) resulted in a ~20% stronger inhibition of Pi uptake. The dose-dependent inhibitory effects were present after 24-hour incubation with both low and high Pi media. Michaelis-Menten kinetics in OK cells identified a ~2.4-fold higher Km for Pi in response to Npt2a inhibition with no significant change in apparent Vmax. Higher PTH concentrations decreased Pi uptake equivalent to the maximal inhibitory effect of Npt2a-I. In vivo, the Npt2a-I induced a dose-dependent increase in urinary Pi excretion in wild-type mice (ED50: ~23 mg/kg), which was completely absent in Npt2a-/- mice, alongside a lack of decrease in plasma Pi. Of note, the Npt2a-I-induced dose-dependent increase in urinary Na+ excretion was still present in Npt2a-/- mice, a response possibly mediated by an off-target acute inhibitory effect of the Npt2a-I on open probability of the epithelial Na+ channel, ENaC, in the cortical collecting duct.

show abstract

Section: F548supporting

confidence: 85%

Section: Methodsmentioning

confidence: 99%

PF-06869206 is a selective inhibitor of renal P_itransport: evidence from in vitro and in vivo studies

Thomas

Xue

Tomilin

et al. 2020

American Journal of Physiology-Renal Physiology

Self Cite

View full text Add to dashboard Cite

show abstract

“…4C and 4D). Previous reports revealed that extracellular or intracellular ATP levels regulate Na + -dependent Pi transport activities in the proximal tubular cells [34,46]. To determine the reason for the reduced Npt2b protein expression, we hypothesized that ATP, which is a substrate of Akp3, is involved in regulating Npt2b expression.…”

Section: Resultsmentioning

confidence: 99%

A Role of Intestinal Alkaline Phosphatase 3 (Akp3) in Inorganic Phosphate Homeostasis

Segawa

Hanazaki

Kirino

et al. 2018

Kidney Blood Press Res

View full text Add to dashboard Cite

Background/Aims: Hyperphosphatemia is a serious complication of late-stage chronic kidney disease (CKD). Intestinal inorganic phosphate (Pi) handling plays an important role in Pi homeostasis in CKD. We investigated whether intestinal alkaline phosphatase 3 (Akp3), the enzyme that hydrolyzes dietary Pi compounds, is a target for the treatment of hyperphosphatemia in CKD. Methods: We investigated Pi homeostasis in Akp3 knockout mice (Akp3^-/-). We also studied the progression of renal failure in an Akp3^-/- mouse adenine treated renal failure model. Plasma, fecal, and urinary Pi and Ca concentration were measured with commercially available kit, and plasma fibroblast growth factor 23, parathyroid hormone, and 1,25(OH)₂D₃ concentration were measured with ELISA. Brush border membrane vesicles were prepared from mouse intestine using the Ca²⁺ precipitation method and used for Pi transport activity and alkaline phosphatase activity. In vivo intestinal Pi absorption was measured with oral ³²P administration. Results: Akp3^-/- mice exhibited reduced intestinal type II sodium-dependent Pi transporter (Npt2b) protein levels and Na-dependent Pi co-transport activity. In addition, plasma active vitamin D levels were significantly increased in Akp3^-/- mice compared with wild-type animals. In the adenine-induced renal failure model, Akp3 gene deletion suppressed hyperphosphatemia. Conclusion: The present findings indicate that intestinal Akp3 deletion affects Na⁺-dependent Pi transport in the small intestine. In the adenine-induced renal failure model, Akp3 is predicted to be a factor contributing to suppression of the plasma Pi concentration.

show abstract

“…In addition to NaPiIIa (e.g. Markovich et al., ; Pfister et al., ; Thomas, Wagner, Biber, & Hernando, ; Villa‐Bellosta & Sorribas, ), adaptation to a low P i concentration in a culture medium also affects NaPiIIc RNA in OK cells (Figure ). In the case of NaPiIIa (Markovich et al., ) and most probably regarding NaPiIIc, the increase in mRNA seems to be attributable to an increased stability of RNA rather than increased transcription.…”

Section: Discussionmentioning

confidence: 99%

Identification and expression analysis of type II and type III P_i transporters in the opossum kidney cell line

Guillén

Caldas

Levi

et al. 2018

Experimental Physiology

View full text Add to dashboard Cite

New Findings What is the central question of this study?The opossum kidney (OK) cell line is the main in vitro model of proximal tubular Pi transport, but it is incomplete because only the NaPiIIa Pi transporter has been identified. What is the main finding and its importance?We have cloned and characterized the Pi transporters NaPiIIc, PiT1 and PiT2 from OK cells and have analysed the relevance of the four transporters to Pi transport. All four transporters are involved in the upregulated Pi transport of cells incubated using a low‐Pi medium, and only PiT1 is not involved in basal transport. Abstract The apical membrane of renal proximal tubular epithelial cells is the main controller of phosphate homeostasis, because it determines the rate of urinary Pi excretion. The opossum kidney (OK) cell line is a good model for studying this function, but only NaPiIIa (NaPi4) has been identified to date as a Pi transporter in this cell line. In this work, we have identified three additional Pi transporters that are present in OK cells: NaPiIIc, PiT1 and PiT2. All three sequences are similar to the corresponding orthologues, but PiT1 is missing the first transmembrane domain. Confluent cells exhibit characteristics of type II Pi transport, which increases with alkalinity and is inhibited by phosphonoformic acid (PFA), and they mainly express NaPiIIa and NaPiIIc, with a low abundance of PiT1 and PiT2. Proliferating cells show a higher expression of PiT1 and PiT2 and a low expression of NaPiIIa and NaPiIIc. Adaptation to a low Pi concentration for 24 h induces the expression of RNA from NaPiIIa and NaPiIIc, which is not prevented by actinomycin D. Small interfering RNA transfections revealed that PiT1 is not necessary for Pi transport, but it is necessary for adaptation to a low Pi, similar to NaPiIIa and PiT2. Our study reveals the complexity of the coordination between the four Pi transporters, the variability of RNA expression according to confluence and the heterogeneous correlation between Pi transport and RNA levels.

show abstract

Adaptation of Opossum Kidney Cells to Luminal Phosphate: Effects of Phosphonoformic Acid and Kinase Inhibitors

Cited by 8 publications

References 48 publications

PF-06869206 is a selective inhibitor of renal P_itransport: evidence from in vitro and in vivo studies

PF-06869206 is a selective inhibitor of renal P_itransport: evidence from in vitro and in vivo studies

A Role of Intestinal Alkaline Phosphatase 3 (Akp3) in Inorganic Phosphate Homeostasis

Identification and expression analysis of type II and type III P_i transporters in the opossum kidney cell line

Contact Info

Product

Resources

About

Adaptation of Opossum Kidney Cells to Luminal Phosphate: Effects of Phosphonoformic Acid and Kinase Inhibitors

Cited by 8 publications

References 48 publications

PF-06869206 is a selective inhibitor of renal Pitransport: evidence from in vitro and in vivo studies

PF-06869206 is a selective inhibitor of renal Pitransport: evidence from in vitro and in vivo studies

A Role of Intestinal Alkaline Phosphatase 3 (Akp3) in Inorganic Phosphate Homeostasis

Identification and expression analysis of type II and type III Pi transporters in the opossum kidney cell line

Contact Info

Product

Resources

About

PF-06869206 is a selective inhibitor of renal P_itransport: evidence from in vitro and in vivo studies

PF-06869206 is a selective inhibitor of renal P_itransport: evidence from in vitro and in vivo studies

Identification and expression analysis of type II and type III P_i transporters in the opossum kidney cell line