In vitro effects of Npt2a inhibition in renal proximal tubule cells

다국어 초록 (Multilingual Abstract)

Elevated plasma phosphate levels are associated with an increased risk of cardiovascular complications and death. The kidneys play an important role in maintaining plasma phosphate levels and patients with chronic kidney disease (CKD) develop hyperphosphatemia. Two renal transport proteins mediate phosphate reabsorption, the sodium‐phosphate cotransporters Npt2a and Npt2c, with Npt2a accounting for 70 to 80% of phosphate reabsorption and Npt2c for the remainder. We recently described that pharmacological inhibition of Npt2a by a selective Npt2a inhibitor (Npt2a‐I, PF‐06869206) induced phosphaturia and reduced plasma phosphate in a mouse models of normal and reduced kidney function (J Am Soc Nephrol. 2019;30(11):2128–2139). The aim of the current study was to determine the in vitro effects of this Npt2a‐I in opossum kidney (OK) cells, a cellular model of renal epithelia where Npt2a is endogenously expressed. 32P radiotracer was used to study the inhibitory effects of Npt2a‐I on phosphate transport. Npt2a‐I dose‐response (0.01 – 100 μM) studies were performed in the cells by incubating different concentrations of Npt2a‐I with 32P for 10 minutes. Npt2a inhibition caused a dose‐dependent decrease in phosphate transport (from 107±9 pmol/well/min in response to control (DMSO, 0.1%) to a maximum inhibitory effect of 33±1 pmol/well/min at 100 μM Npt2a‐I) showing a half maximal inhibitory concentration (IC50) of ~3 μM. Approximately 70% of phosphate transport in OK cells was inhibited by the highest dose of Npt2a‐I (100 μM); as comparison, the nonselective Npt2 inhibitor phosphonoformate (PFA, 30 mM) inhibited ~90% of phosphate transport. The kinetics of transport inhibition by Npt2a‐I were studied using phosphate concentrations varying from 50 – 400 μM with or without Npt2a‐I (3 μM). Npt2a‐I increased the Michaelis‐Menten constant, Km (506±124 vs 210±22 mM) for phosphate; in contrast, Vmax was unaffected (348±31 vs 300±54 pmol/min/well), indicating competitive inhibition. Preincubation of OK cells with 3 μM Npt2a‐I for 1, 2 or 3 hours caused a significant reduction in phosphate transport compared to control (29±1 vs 40±1, 28±1 vs 37±1 and 30±1 vs 37±1 pmol/min/mg protein, respectively); whereas 24 hour preincubation increased phosphate transport significantly (49±3 vs 39±2 pmol/min/mg protein). Twenty‐four hour of incubation with vehicle or Npt2a‐I (30 μM) did not show cytotoxic effects (5.1±0.9 and 4.7±0.3% dead cells). In summary, our studies demonstrate that in vitro Npt2a inhibition causes a dose‐dependent reduction in phosphate transport in OK cells without affecting their viability. Our kinetic data imply the compound acts as a competitive Npt2a inhibitor.
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases grant 1R01DK110621 (to Dr. Rieg) and American Heart Association Transformational Research Award 19TPA34850116 (to Dr. Rieg). Dr. Thomas was supported by an American Heart Association postdoctoral fellowship (19POST34400026) and Mr. Xue by a predoctoral fellowship (18PRE33990236).