area of basolateral cell membrane  (Thermo Fisher)

Journal: Toxins

Article Title: The Influence of OAT1 Density and Functionality on Indoxyl Sulfate Transport in the Human Proximal Tubule: An Integrated Computational and In Vitro Study

doi: 10.3390/toxins13100674

Figure Lengend Snippet: Simulations of the standard model using the fitted parameters of the parameter-fitting model. ( a ) A 17.5 h extended simulation of the standard model to show the trends of 25 µM indoxyl sulfate (IS) complexed with albumin, unbound IS in the cell monolayer, and unbound IS in the dialysate. ( b ) Sensitivity analysis of the standard model to investigate the influence of a 20% decrease in transporter density, uptake and dissociation rate, albumin concentration, and albumin binding rate to IS on IS transport to the dialysate. ( c ) Step variations of initial albumin concentration (1 × 10 −2 , 1 × 10 0 and 1 × 10 2 mM) in the blood compartment. ( d ) Step variations of unbound OAT1 density (1.15 × 10 5 , 1.15 × 10 7 , 1.15 × 10 9 molecules/µm 2 ) along the basolateral cell membrane. ( e ) Step variations to the dissociation rate (4.18 × 10 −6 , 4.18 × 10 −4 , 4.18 × 10 −2 s −1 ) of IS from OAT1 into the cell monolayer. ( f ) Step variations to the uptake rate of IS (1.75 × 10 −7 , 1.75 × 10 −5 , 1.75 × 10 −3 s −1 µM −1 ) by OAT1 in the blood compartment.

Article Snippet: Area of Basolateral Cell Membrane [cm 2 ] , Size membrane , 0.32 , , Thermo Scientific.

Techniques: Concentration Assay, Binding Assay, Membrane

Journal: Toxins

Article Title: The Influence of OAT1 Density and Functionality on Indoxyl Sulfate Transport in the Human Proximal Tubule: An Integrated Computational and In Vitro Study

doi: 10.3390/toxins13100674

Figure Lengend Snippet: Simulations of the uremic conditions using I S B , t = 0 = 180 µ M ; f1 = 1 × 10 −5 and f2 = 0.0.44. ( a ) The complete dynamic profile of the IS reacting species in the simulations until complete removal (84 h time period). The right axis plots intracellular IS (IS-Cell) concentration. While the left axis plots the IS–albumin complex (Complex) and IS concentration in the basolateral and apical compartments. ( b ) Sensitivity analysis comparing the uremic ( I S B , t = 0 = 180 µM) and physiological ( I S B , t = 0 = 2.5 µM) IS concentrations with albumin conformational changes (f1 = 1 × 10 −5 , f2 = 0.044 and I S B , t = 0 = 180 µM) by reducing the individual parameters (uptake rate, dissociation rate, transporter density, albumin binding, and albumin concentration) by 20%.

Article Snippet: Area of Basolateral Cell Membrane [cm 2 ] , Size membrane , 0.32 , , Thermo Scientific.

Techniques: Concentration Assay, Binding Assay

Journal: Toxins

Article Title: The Influence of OAT1 Density and Functionality on Indoxyl Sulfate Transport in the Human Proximal Tubule: An Integrated Computational and In Vitro Study

doi: 10.3390/toxins13100674

Figure Lengend Snippet: Main parameters for the parameter fitting and standard Models. Note that we modeled various albumin condiions in mM albumin (standard model— Section 5.4 , physiological condition and uremic condition— Section 5.6 ) or without albumin (for the parameter-fitting model since the experiments were performed in the absence of albumin— Section 5.1 ).

Article Snippet: Area of Basolateral Cell Membrane [cm 2 ] , Size membrane , 0.32 , , Thermo Scientific.

Techniques: Binding Assay, Membrane

Journal: Toxins

Article Title: The Influence of OAT1 Density and Functionality on Indoxyl Sulfate Transport in the Human Proximal Tubule: An Integrated Computational and In Vitro Study

doi: 10.3390/toxins13100674

Figure Lengend Snippet: ( a ) Schematic of the parameter-fitting model based on data from IS cellular uptake experiments with four reacting species: (1) free indoxyl sulfate, I S W e l l ; (2) unbound OAT 1 ; (3) IS bound to OAT 1 , I S O A T 1 ; (4) IS transported to the proximal tubule monolayer, I S C e l l . IS binds to OAT1 on the ciPTECs-OAT1 basolateral membrane at a rate k f I S , U p t a k e and dissociates from the cell monolayer at a rate k f D i s s o c i a t i o n . The IS is transported back to the well at the efflux rate of the breast cancer-resistant protein (BCRP) efflux pump. ( b ) Schematic of the standard protein-bound uremic toxin model. The model uses seven reacting species: (1) free indoxyl sulfate, I S B ; (2) human serum albumin; (3) IS bound to albumin, Complex; (4) unbound OAT 1 ; (5) IS bound to OAT 1 , I S O A T 1 ; (6) IS transported to the proximal tubule monolayer, I S C e l l ; (7) IS excreted to the dialysate/lumen, I S D . The reactions occur within three compartments: the blood, the proximal tubule epithelial cell monolayer, and the dialysate. The basolateral membrane separates the blood and the cell monolayer, whereas the apical membrane separates the cell monolayer and the dialysate.

Article Snippet: Area of Basolateral Cell Membrane [cm 2 ] , Size membrane , 0.32 , , Thermo Scientific.

Techniques: Membrane

Journal: The Journal of Biological Chemistry

Article Title: Angiotensin II increases activity of the ClC-K2 Cl − channel in collecting duct intercalated cells by stimulating production of reactive oxygen species

doi: 10.1016/j.jbc.2021.100347

Figure Lengend Snippet: Ang II increases basolateral chloride currents in intercalated cells of the collecting duct. A , representative macroscopic currents in individual principal cells in response to voltage steps from −90 to +60 from the holding potential of −60 mV in the control ( black ) and following treatment with Ang II (500 nM) for 3 min ( gray ). A micrograph of a typical isolated collecting duct shown on top . The expression of AQP2, a marker of principal cells (highlighted with yellow arrows ), is shown with pseudocolor green . Nuclear marker DAPI is shown with pseudocolor blue . The scale bar represents 70 μM. B , current–voltage (I–V) relations of the basolateral K + -selective conductance obtained from voltage step protocols as shown in A in the control ( black ) and upon treatment with Ang II ( gray ). The number of individual recordings is shown. Measurements were done from at least three different mice. Both SEM ( smaller bars ) and SD ( larger bars ) are shown for each measured value. C , representative macroscopic currents in individual intercalated cells in response to voltage steps from −90 to +60 from the holding potential of −60 mV in the control ( black ) and following treatment with Ang II (500 nM) for 3 min ( gray ). A micrograph of a typical isolated collecting duct shown on top. The expression of ClC-K2, a marker of intercalated cells (highlighted with white arrows ), is shown with pseudocolor red . Nuclear marker DAPI is shown with pseudocolor blue . The scale bar represents 70 μM. D , current–voltage relations of the basolateral Cl − -selective conductance obtained from voltage step protocols as shown in A in the control ( black ) and upon treatment with Ang II ( gray ). The number of individual recordings is shown. Measurements were done from at least three different mice. Both SEM ( smaller bars ) and SD ( larger bars ) are shown for each measured value. ∗ - significant change ( p < 0.05) versus respective control (one-way ANOVA). DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: To dissolve the basal lamina and to get direct access to the basolateral membrane, isolated sectors were further incubated in the Ringer solution containing 0.8 mg/ml collagenase type I (Alfa Aesar) and 5 mg/ml of dispase II (Roche Diagnostics) for 20 min at 37 °C followed by extensive washout.

Techniques: Control, Isolation, Expressing, Marker

Journal: The Journal of Biological Chemistry

Article Title: Angiotensin II increases activity of the ClC-K2 Cl − channel in collecting duct intercalated cells by stimulating production of reactive oxygen species

doi: 10.1016/j.jbc.2021.100347

Figure Lengend Snippet: Ang II does not affect the activity of the basolateral K ir 4.1/5.1 channel in principal cells. A , representative continuous current trace from a cell-attached patch monitoring activity of the basolateral 40 pS K ir 4.1/5.1 potassium channels in a principal cell in a freshly isolated collecting duct at the baseline, upon application of 500 nM Ang II (shown with a line on top ) and following washout with control medium. The patch was clamped to −V p = −40 mV. Areas (1, control) and (2, Ang II) are shown below at an expanded timescale; “c” denotes closed nonconducting state. B , summary graph of changes in K ir 4.1/5.1 open probability ( P o ) upon treatment with Ang II from paired patch clamp experiments similar to that shown in ( A ). Collecting ducts from at least three different mice were used.

Article Snippet: To dissolve the basal lamina and to get direct access to the basolateral membrane, isolated sectors were further incubated in the Ringer solution containing 0.8 mg/ml collagenase type I (Alfa Aesar) and 5 mg/ml of dispase II (Roche Diagnostics) for 20 min at 37 °C followed by extensive washout.

Techniques: Activity Assay, Isolation, Control, Patch Clamp

Journal: The Journal of Biological Chemistry

Article Title: Angiotensin II increases activity of the ClC-K2 Cl − channel in collecting duct intercalated cells by stimulating production of reactive oxygen species

doi: 10.1016/j.jbc.2021.100347

Figure Lengend Snippet: Ang II stimulates activity of ClC-K2 channel in a dose-dependent manner. A , representative continuous current trace from a cell-attached patch monitoring activity of basolateral 10 pS ClC-K2 chloride channels in an intercalated cell in a freshly isolated collecting duct in the control, upon application of 500 nM Ang II (shown with a line on top ) and following washout with control medium. The patch was clamped to −V p = −60 mV; “c” denotes closed nonconducting state. Areas (1, control) and (2, Ang II) are shown below at an expanded timescale. B , summary graph of changes in ClC-K2 open probability ( P o ) upon treatment with 500 nM Ang II from paired patch clamp experiments similar to that shown in ( A ). C , summary graph of average changes in ClC-K2 P o in individual cells upon application of different Ang II concentrations. ∗ - significant increase ( p < 0.05) versus control (one-way ANOVA). Collecting ducts from at least three different mice were used for each set of experiments.

Article Snippet: To dissolve the basal lamina and to get direct access to the basolateral membrane, isolated sectors were further incubated in the Ringer solution containing 0.8 mg/ml collagenase type I (Alfa Aesar) and 5 mg/ml of dispase II (Roche Diagnostics) for 20 min at 37 °C followed by extensive washout.

Techniques: Activity Assay, Isolation, Control, Patch Clamp

Journal: The Journal of Biological Chemistry

Article Title: Angiotensin II increases activity of the ClC-K2 Cl − channel in collecting duct intercalated cells by stimulating production of reactive oxygen species

doi: 10.1016/j.jbc.2021.100347

Figure Lengend Snippet: Ang II increases ClC-K2 activity in intercalated cells by acting on AT 1 receptors. A , representative continuous current trace from a cell-attached patch monitoring activity of basolateral ClC-K2 chloride channels in an intercalated cell of a freshly isolated collecting duct in the control, upon treatment with AT 1 receptor blocker losartan (1 μM, gray line ), and Ang II (500 nM, black line ) in the continued presence of the antagonist. The patch was clamped to −V p = −60 mV; “c” denotes closed nonconducting state. Areas (1, control) and (2, Ang II + losartan) are shown below at an expanded timescale. B , summary graph of changes in ClC-K2 open probability ( P o ) upon treatment with losartan and following Ang II in paired patch clamp experiments similar to that shown in ( A ). C , summary graph of changes in ClC-K2 P o in the control, during treatment with AT 2 receptor agonist, CGP42112 (100 nM for 3 min), and following washout with control medium. Collecting ducts from at least three different mice were used for each set of experiments.

Article Snippet: To dissolve the basal lamina and to get direct access to the basolateral membrane, isolated sectors were further incubated in the Ringer solution containing 0.8 mg/ml collagenase type I (Alfa Aesar) and 5 mg/ml of dispase II (Roche Diagnostics) for 20 min at 37 °C followed by extensive washout.

Techniques: Activity Assay, Isolation, Control, Patch Clamp

Journal: The Journal of Biological Chemistry

Article Title: Angiotensin II increases activity of the ClC-K2 Cl − channel in collecting duct intercalated cells by stimulating production of reactive oxygen species

doi: 10.1016/j.jbc.2021.100347

Figure Lengend Snippet: Inhibition of PLC, PI3-K, and phospholipase A2 signaling cascades does not affect regulation of ClC-K2 activity by Ang II. A , representative continuous current trace from a cell-attached patch monitoring activity of basolateral ClC-K2 chloride channels in an intercalated cell of a freshly isolated collecting duct in the control, upon treatment with PLC inhibitor, U73122 (10 μM, gray line), Angiotensin II (500 nM, black line ) in the continued presence of the blocker, and following washout with the control medium. The patch was clamped to −V p = −60 mV; “c” denotes closed nonconducting state. Areas (1, control) and (2, Ang II + U73122) are shown below at an expanded timescale. Summary graphs of changes in ClC-K2 open probability ( P o ) upon pretreatment with PLC blocker, 10 μM U73122 ( B ), PI3-K blocker, 20 μM LY294002 ( C ), phospholipase A2 blocker, 30 μM AACOCF 3 ( D ); Ang II application in the continued presence of the respective antagonist, and following washout with control medium, as similarly shown in paired patch clamp experiment in ( A ). ∗ - significant increase ( p < 0.05) versus pretreatment with respective blocker (one-way ANOVA); ‡ - significant decrease ( p < 0.05) versus control (one-way ANOVA). Collecting ducts from at least three different mice were used for each set of experiments. PLC, phospholipase C.

Article Snippet: To dissolve the basal lamina and to get direct access to the basolateral membrane, isolated sectors were further incubated in the Ringer solution containing 0.8 mg/ml collagenase type I (Alfa Aesar) and 5 mg/ml of dispase II (Roche Diagnostics) for 20 min at 37 °C followed by extensive washout.

Techniques: Inhibition, Activity Assay, Isolation, Control, Patch Clamp

Journal: The Journal of Biological Chemistry

Article Title: Angiotensin II increases activity of the ClC-K2 Cl − channel in collecting duct intercalated cells by stimulating production of reactive oxygen species

doi: 10.1016/j.jbc.2021.100347

Figure Lengend Snippet: Ang II increases ClC-K2 activity in intercalated cells in a NOX-dependent manner. A , representative continuous current trace from a cell-attached patch monitoring activity of basolateral ClC-K2 chloride channels in an intercalated cell of a freshly isolated collecting duct in the control, upon treatment with NOX inhibitor, apocynin (100 μM, gray line), Angiotensin II (500 nM, black line ) in the continued presence of the blocker, and following washout with the control medium. The patch was clamped to −V p = −60 mV; “c” denotes closed nonconducting state. Areas (1, control) and (2, Ang II + apocynin) are shown below at an expanded timescale. B , summary graph of changes in ClC-K2 open probability ( P o ) upon treatment with apocynin, Ang II in the continued presence of the blocker, and following washout with control medium in paired patch clamp experiments similar to that shown in ( A ). Collecting ducts from at least three different mice were used.

Article Snippet: To dissolve the basal lamina and to get direct access to the basolateral membrane, isolated sectors were further incubated in the Ringer solution containing 0.8 mg/ml collagenase type I (Alfa Aesar) and 5 mg/ml of dispase II (Roche Diagnostics) for 20 min at 37 °C followed by extensive washout.

Techniques: Activity Assay, Isolation, Control, Patch Clamp

Journal: The Journal of Biological Chemistry

Article Title: Angiotensin II increases activity of the ClC-K2 Cl − channel in collecting duct intercalated cells by stimulating production of reactive oxygen species

doi: 10.1016/j.jbc.2021.100347

Figure Lengend Snippet: Principal scheme of Ang II actions on the basolateral conductance in intercalated and principal cells of the collecting duct. Green arrows represent stimulatory actions and red lines demonstrate successful interruption of the stimulatory pathway with pharmacology. AT 1 R, angiotensin receptor type 1; PLA 2 , phospholipase A2; PLC, phospholipase C; NOX, NADPH oxidase; PI3K, phosphoinositide 3 kinase; ROS, reactive oxygen species.

Article Snippet: To dissolve the basal lamina and to get direct access to the basolateral membrane, isolated sectors were further incubated in the Ringer solution containing 0.8 mg/ml collagenase type I (Alfa Aesar) and 5 mg/ml of dispase II (Roche Diagnostics) for 20 min at 37 °C followed by extensive washout.

Techniques: