Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: Gpr116 is highly expressed in kidney and is localized to V-ATPase expressing ICs of the collecting duct. (A) Gpr116 mRNA expression as measured by qRT-PCR in various tissues including lung and kidney. SP-C and AQP2 served as positive controls of lung and kidney mRNA. Gene expression is normalized to Gapdh. Cycle threshold of 35 approximately corresponds with 0.001 on the y axis. (B) Western blot analysis of Gpr116 expression in various tissues as well as stably transfected HEK293 cells with mouse (mGpr116) or human (hGpr116) Gpr116. Expected molecular mass for Gpr116 is ∼150 kDa. (C) Diagram of the murine outer medullary collecting duct (OMCD) showing water-transporting principal cells (PC) and acid-secreting A-IC. Proton secretion is coupled to bicarbonate reabsorption throughout the nephron. (D) Representative immunofluorescence images demonstrating colocalization of Gpr116 with V-ATPase in mouse collecting ducts. Similarly, Gpr116 is interspersed among AQP2 expressing cells in mouse collecting ducts. (Scale bars, 20 μm.)

Article Snippet: Primary antibody targeting AQP2 was a kind gift of Paul Welling, The Johns Hopkins University, Baltimore, MD ( 56 ), and primary antibody targeting pendrin was a kind gift of Susan Wall, Emory University, Atlanta, GA ( 57 ).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Western Blot, Stable Transfection, Transfection, Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: Targeted deletion of Gpr116 in kidney tubules results in loss of Gpr116 in collecting ducts. (A) Representative immunofluorescence images demonstrating localization of Gpr116-expressing cells among AQP2 expressing cells in WT mouse collecting ducts. (B) Representative immunofluorescence images demonstrating loss of Gpr116-expressing cells in mouse collecting ducts after targeted deletion with KSP-Cre/Lox system. (Scale bars, 20 μm.)

Article Snippet: Primary antibody targeting AQP2 was a kind gift of Paul Welling, The Johns Hopkins University, Baltimore, MD ( 56 ), and primary antibody targeting pendrin was a kind gift of Susan Wall, Emory University, Atlanta, GA ( 57 ).

Techniques: Immunofluorescence, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: KO mice do not have more A-ICs than WT mice. (A) Representative images demonstrating AQP2 (green) labeling of principal cells and AE1 (red) labeling of ICs from mice drinking control water and water with NH4Cl. (Scale bars, 20 μm.) (B, Left) Quantification of AE1+ cells per 100 DAPI+ cells. For each kidney section, quantification was performed on whole medulla. (Right) Quantification of AQP2+ cells per 100 DAPI+ cells. For B, bars are mean ± SEM. n = 6 kidneys per group. Solid bars are 0.5% sucrose water, hatched bars are 0.5% sucrose + 280 mM NH4Cl water.

Article Snippet: Primary antibody targeting AQP2 was a kind gift of Paul Welling, The Johns Hopkins University, Baltimore, MD ( 56 ), and primary antibody targeting pendrin was a kind gift of Susan Wall, Emory University, Atlanta, GA ( 57 ).

Techniques: Labeling, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: mRNAs of V-ATPase subunits are similar in WT and KO mice. qPCR analysis of AQP2, SLC4A1 (AE1), V-ATPase subunits ATP6V1B1, ATP6V0D2, ATP6V1G3, AQP6, and CLCN5 mRNAs are not statistically different in WT and KO kidney samples. V-ATPase subunits ATP6V0D2 and ATP6V1G3 were previously shown to be unique transcriptional markers for A-ICs in the kidney (18). AQP6 and CLCN5 encode for proteins that colocalize with V-ATPase in subapical vesicles (59). Bars are mean ± SEM. n = 4 WT kidneys, 3 KO kidneys. Cycle threshold of 35 approximately corresponds with 0.001 on the y axis.

Article Snippet: Primary antibody targeting AQP2 was a kind gift of Paul Welling, The Johns Hopkins University, Baltimore, MD ( 56 ), and primary antibody targeting pendrin was a kind gift of Susan Wall, Emory University, Atlanta, GA ( 57 ).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: Gpr116 activation in A-ICs inhibits pHi recovery. (A) Representative pseudocolor images (blue, acidic; red, alkali) of intracellular pH in a split opened collecting duct loaded with pH-sensitive dye BCECF at the baseline (1), upon application of 40 mM NH4Cl (2), immediately after NH4Cl removal (3), and recovery toward the baseline pHi values (4). Confocal micrograph of the same split-opened CD probed with anti-AQP2 (pseudocolor red) is shown on the right. Examples of AQP2− ICs are depicted with white arrows. Nuclear DAPI staining is shown in pseudocolor blue. Magnification: 40×. (B) Summary graph comparing the time course of pHi changes in control (black) and p16 pretreated (100 µM for 40 min, red) ICs. Trace shows mean ± SEM for 65 control cells and 69 p16-treated cells. Each experimental condition is a summary from four collecting ducts from four different mice. Application of the NH4Cl pulse is designated by the black bar above the trace. The time points shown in A are marked as 1 to 4. (C) Summary graph of H+ extrusion rate from ICs from the control and after pretreatment with p16 shown in B. The rate was calculated as a linear slope of pHi recovery after 40 mM NH4Cl application. Statistical analysis performed using Mann–Whitney test. *P < 0.05. Control vs. p16 *P < 0.0001.

Article Snippet: Primary antibody targeting AQP2 was a kind gift of Paul Welling, The Johns Hopkins University, Baltimore, MD ( 56 ), and primary antibody targeting pendrin was a kind gift of Susan Wall, Emory University, Atlanta, GA ( 57 ).

Techniques: Activation Assay, Staining, Control, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: Antibodies used in the study

Article Snippet: Primary antibody targeting AQP2 was a kind gift of Paul Welling, The Johns Hopkins University, Baltimore, MD ( 56 ), and primary antibody targeting pendrin was a kind gift of Susan Wall, Emory University, Atlanta, GA ( 57 ).

Techniques: Diagnostic Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

doi: 10.1073/pnas.2007620117

Figure Lengend Snippet: TaqMan real-time PCR assays used

Article Snippet: Primary antibody targeting AQP2 was a kind gift of Paul Welling, The Johns Hopkins University, Baltimore, MD ( 56 ), and primary antibody targeting pendrin was a kind gift of Susan Wall, Emory University, Atlanta, GA ( 57 ).

Techniques: Real-time Polymerase Chain Reaction