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Contribution of the organic anion transporter OAT2 to the renal active tubular secretion of creatinine and mechanism for serum creatinine elevations caused by cobicistat.

Lepist EI, Zhang X, Hao J, Huang J, Kosaka A, Birkus G, Murray BP, Bannister R, Cihlar T, Huang Y, Ray AS - Kidney Int. (2014)

Bottom Line: This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects.The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine.Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 μM for creatinine transport.

View Article: PubMed Central - PubMed

Affiliation: Gilead Sciences, Foster City, California, USA.

ABSTRACT
Many xenobiotics including the pharmacoenhancer cobicistat increase serum creatinine by inhibiting its renal active tubular secretion without affecting the glomerular filtration rate. This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects. The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine. At physiologic creatinine concentrations, the specific activity of OAT2 transport was over twofold higher than OCT2 or OCT3, establishing OAT2 as a likely relevant creatinine transporter and further challenging the traditional view that creatinine is solely transported by a cationic pathway. The apical multidrug and toxin extrusion transporters MATE1 and MATE2-K demonstrated low-affinity and high-capacity transport. All drugs known to affect creatinine inhibited OCT2 and MATE1. Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 μM for creatinine transport. Trimethoprim potently inhibited MATE2-K, whereas dolutegravir preferentially inhibited OCT2. Cimetidine was unique, inhibiting all transporters that interact with creatinine. Thus, the clinical observation of elevated serum creatinine in patients taking cobicistat is likely a result of OCT2 transport, facilitating intracellular accumulation, and MATE1 inhibition.

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Uptake transport of cobicistat by renal transporters. (a) The uptake of cobicistat (1 μmol/l) was assessed in fresh isolated primary proximal tubule cells in the presence of a model cationic transport inhibitor cimetidine (1500 μmol/l) and anionic transport inhibitor probenecid (90 μmol/l). (b) The cimetidine-sensitive uptake of the model cationic substrate tetraethylammonium or probenecid-sensitive anionic substrate esterone-3-sulfate was determined by establishing transporter expression in the primary proximal tubule cells. The effect of OCT2 or OAT3 expression on the intracellular accumulation of cobicistat (1 μmol/l) was assessed in transfected cell lines. (c) The effect of inhibitors on levels of cobicistat in parental (nontransfected) or Madin–Darby canine kidney II (MDCKII) cells transfected with the organic cation transporter OCT2 were assessed in the presence or absence of the OCT2 inhibitors cimetidine (1500 μmol/l) or quinidine (200 μmol/l). (d) The inhibitor-sensitive accumulation of the known OCT2 substrate tetraethylammonium was determined, establishing appropriate OCT2 expression. (e) The effect of inhibitors on levels of cobicistat in human embryonic kidney 293 (HEK293) cells either mock-transfected or transfected with the organic anion transporter OAT3 were assessed in the presence or absence of the OAT3 inhibitors diclofenac or probenecid both incubated at 90 μmol/l. (f) The inhibitor-sensitive accumulation of the known OAT3 substrate esterone-3-sulfate was determined, establishing appropriate OAT3 expression. Data represent the average±s.d. of three independent assays performed in triplicate. The statistical significance of differences between conditions was assessed using the Student's unpaired and two-tailed t-test assuming equal variance (*P<0.05; **P<0.005; ***P<0.001; and ****P<0.0001).
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fig3: Uptake transport of cobicistat by renal transporters. (a) The uptake of cobicistat (1 μmol/l) was assessed in fresh isolated primary proximal tubule cells in the presence of a model cationic transport inhibitor cimetidine (1500 μmol/l) and anionic transport inhibitor probenecid (90 μmol/l). (b) The cimetidine-sensitive uptake of the model cationic substrate tetraethylammonium or probenecid-sensitive anionic substrate esterone-3-sulfate was determined by establishing transporter expression in the primary proximal tubule cells. The effect of OCT2 or OAT3 expression on the intracellular accumulation of cobicistat (1 μmol/l) was assessed in transfected cell lines. (c) The effect of inhibitors on levels of cobicistat in parental (nontransfected) or Madin–Darby canine kidney II (MDCKII) cells transfected with the organic cation transporter OCT2 were assessed in the presence or absence of the OCT2 inhibitors cimetidine (1500 μmol/l) or quinidine (200 μmol/l). (d) The inhibitor-sensitive accumulation of the known OCT2 substrate tetraethylammonium was determined, establishing appropriate OCT2 expression. (e) The effect of inhibitors on levels of cobicistat in human embryonic kidney 293 (HEK293) cells either mock-transfected or transfected with the organic anion transporter OAT3 were assessed in the presence or absence of the OAT3 inhibitors diclofenac or probenecid both incubated at 90 μmol/l. (f) The inhibitor-sensitive accumulation of the known OAT3 substrate esterone-3-sulfate was determined, establishing appropriate OAT3 expression. Data represent the average±s.d. of three independent assays performed in triplicate. The statistical significance of differences between conditions was assessed using the Student's unpaired and two-tailed t-test assuming equal variance (*P<0.05; **P<0.005; ***P<0.001; and ****P<0.0001).

Mentions: To explore the possibility that cobicistat accumulation in proximal tubule cells has a role in serum creatinine elevations observed clinically, we assessed the uptake of cobicistat into fresh human primary proximal tubule cells and cell lines overexpressing individual transporters. Assessing cobicistat accumulation in cells was technically challenging because of the high levels of nonspecific membrane association observed for this lipophilic drug. Addition of protein to the incubation and isolation of cells by spinning through oil reduced the background to allow observation of transporter-dependent uptake (see Materials and Methods). Although transporter activity is variable and progressively declining after isolation, evidence for transporter-dependent uptake of cobicistat was observed in freshly isolated primary proximal tubule cells based on small but reproducible decreases in accumulation in the presence of the transport inhibitors cimetidine or probenecid (Figure 3a). To better characterize the molecular mechanism for cobicistat accumulation in proximal tubule cells, uptake studies were completed in cells overexpressing individual basolateral uptake transporters. OCT2 and OAT3 were selected on the basis of the apparent sensitivity of cobicistat accumulation to cimetidine and probenecid in proximal tubule cells and evidence for some molecular interaction provided by the weak inhibition of these transporters by cobicistat reported here and elsewhere.28 A 2.9-fold increase in cobicistat accumulation was observed in cells transfected with OCT2 (Figure 3c). Uptake in OCT2-expressing cells was reduced to that of nontransfected cells in the presence of OCT2 inhibitors. Although quinidine caused a significant increase in cobicistat accumulation in nontransfected cells, likely related to the inhibition of p-glycoprotein-mediated cobicistat efflux, it completely inhibited the uptake transport in OCT2-expressing cells, reducing accumulation to levels observed in nontransfected cells in the absence of inhibitor. In contrast, no inhibitor-sensitive accumulation of cobicistat was observed in cells transfected with OAT3 (Figure 3e). Transporter expression was established in the different systems with the use of model substrates (Figure 3b, d, and f).


Contribution of the organic anion transporter OAT2 to the renal active tubular secretion of creatinine and mechanism for serum creatinine elevations caused by cobicistat.

Lepist EI, Zhang X, Hao J, Huang J, Kosaka A, Birkus G, Murray BP, Bannister R, Cihlar T, Huang Y, Ray AS - Kidney Int. (2014)

Uptake transport of cobicistat by renal transporters. (a) The uptake of cobicistat (1 μmol/l) was assessed in fresh isolated primary proximal tubule cells in the presence of a model cationic transport inhibitor cimetidine (1500 μmol/l) and anionic transport inhibitor probenecid (90 μmol/l). (b) The cimetidine-sensitive uptake of the model cationic substrate tetraethylammonium or probenecid-sensitive anionic substrate esterone-3-sulfate was determined by establishing transporter expression in the primary proximal tubule cells. The effect of OCT2 or OAT3 expression on the intracellular accumulation of cobicistat (1 μmol/l) was assessed in transfected cell lines. (c) The effect of inhibitors on levels of cobicistat in parental (nontransfected) or Madin–Darby canine kidney II (MDCKII) cells transfected with the organic cation transporter OCT2 were assessed in the presence or absence of the OCT2 inhibitors cimetidine (1500 μmol/l) or quinidine (200 μmol/l). (d) The inhibitor-sensitive accumulation of the known OCT2 substrate tetraethylammonium was determined, establishing appropriate OCT2 expression. (e) The effect of inhibitors on levels of cobicistat in human embryonic kidney 293 (HEK293) cells either mock-transfected or transfected with the organic anion transporter OAT3 were assessed in the presence or absence of the OAT3 inhibitors diclofenac or probenecid both incubated at 90 μmol/l. (f) The inhibitor-sensitive accumulation of the known OAT3 substrate esterone-3-sulfate was determined, establishing appropriate OAT3 expression. Data represent the average±s.d. of three independent assays performed in triplicate. The statistical significance of differences between conditions was assessed using the Student's unpaired and two-tailed t-test assuming equal variance (*P<0.05; **P<0.005; ***P<0.001; and ****P<0.0001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4120670&req=5

fig3: Uptake transport of cobicistat by renal transporters. (a) The uptake of cobicistat (1 μmol/l) was assessed in fresh isolated primary proximal tubule cells in the presence of a model cationic transport inhibitor cimetidine (1500 μmol/l) and anionic transport inhibitor probenecid (90 μmol/l). (b) The cimetidine-sensitive uptake of the model cationic substrate tetraethylammonium or probenecid-sensitive anionic substrate esterone-3-sulfate was determined by establishing transporter expression in the primary proximal tubule cells. The effect of OCT2 or OAT3 expression on the intracellular accumulation of cobicistat (1 μmol/l) was assessed in transfected cell lines. (c) The effect of inhibitors on levels of cobicistat in parental (nontransfected) or Madin–Darby canine kidney II (MDCKII) cells transfected with the organic cation transporter OCT2 were assessed in the presence or absence of the OCT2 inhibitors cimetidine (1500 μmol/l) or quinidine (200 μmol/l). (d) The inhibitor-sensitive accumulation of the known OCT2 substrate tetraethylammonium was determined, establishing appropriate OCT2 expression. (e) The effect of inhibitors on levels of cobicistat in human embryonic kidney 293 (HEK293) cells either mock-transfected or transfected with the organic anion transporter OAT3 were assessed in the presence or absence of the OAT3 inhibitors diclofenac or probenecid both incubated at 90 μmol/l. (f) The inhibitor-sensitive accumulation of the known OAT3 substrate esterone-3-sulfate was determined, establishing appropriate OAT3 expression. Data represent the average±s.d. of three independent assays performed in triplicate. The statistical significance of differences between conditions was assessed using the Student's unpaired and two-tailed t-test assuming equal variance (*P<0.05; **P<0.005; ***P<0.001; and ****P<0.0001).
Mentions: To explore the possibility that cobicistat accumulation in proximal tubule cells has a role in serum creatinine elevations observed clinically, we assessed the uptake of cobicistat into fresh human primary proximal tubule cells and cell lines overexpressing individual transporters. Assessing cobicistat accumulation in cells was technically challenging because of the high levels of nonspecific membrane association observed for this lipophilic drug. Addition of protein to the incubation and isolation of cells by spinning through oil reduced the background to allow observation of transporter-dependent uptake (see Materials and Methods). Although transporter activity is variable and progressively declining after isolation, evidence for transporter-dependent uptake of cobicistat was observed in freshly isolated primary proximal tubule cells based on small but reproducible decreases in accumulation in the presence of the transport inhibitors cimetidine or probenecid (Figure 3a). To better characterize the molecular mechanism for cobicistat accumulation in proximal tubule cells, uptake studies were completed in cells overexpressing individual basolateral uptake transporters. OCT2 and OAT3 were selected on the basis of the apparent sensitivity of cobicistat accumulation to cimetidine and probenecid in proximal tubule cells and evidence for some molecular interaction provided by the weak inhibition of these transporters by cobicistat reported here and elsewhere.28 A 2.9-fold increase in cobicistat accumulation was observed in cells transfected with OCT2 (Figure 3c). Uptake in OCT2-expressing cells was reduced to that of nontransfected cells in the presence of OCT2 inhibitors. Although quinidine caused a significant increase in cobicistat accumulation in nontransfected cells, likely related to the inhibition of p-glycoprotein-mediated cobicistat efflux, it completely inhibited the uptake transport in OCT2-expressing cells, reducing accumulation to levels observed in nontransfected cells in the absence of inhibitor. In contrast, no inhibitor-sensitive accumulation of cobicistat was observed in cells transfected with OAT3 (Figure 3e). Transporter expression was established in the different systems with the use of model substrates (Figure 3b, d, and f).

Bottom Line: This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects.The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine.Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 μM for creatinine transport.

View Article: PubMed Central - PubMed

Affiliation: Gilead Sciences, Foster City, California, USA.

ABSTRACT
Many xenobiotics including the pharmacoenhancer cobicistat increase serum creatinine by inhibiting its renal active tubular secretion without affecting the glomerular filtration rate. This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects. The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine. At physiologic creatinine concentrations, the specific activity of OAT2 transport was over twofold higher than OCT2 or OCT3, establishing OAT2 as a likely relevant creatinine transporter and further challenging the traditional view that creatinine is solely transported by a cationic pathway. The apical multidrug and toxin extrusion transporters MATE1 and MATE2-K demonstrated low-affinity and high-capacity transport. All drugs known to affect creatinine inhibited OCT2 and MATE1. Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 μM for creatinine transport. Trimethoprim potently inhibited MATE2-K, whereas dolutegravir preferentially inhibited OCT2. Cimetidine was unique, inhibiting all transporters that interact with creatinine. Thus, the clinical observation of elevated serum creatinine in patients taking cobicistat is likely a result of OCT2 transport, facilitating intracellular accumulation, and MATE1 inhibition.

Show MeSH
Related in: MedlinePlus