Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Vesicles and tubules internalize CT to a perinuclear endosomal compartment. (A) CT traffics in vesicles (arrows) and tubules (arrowheads) from the plasma membrane toward the perinuclear endosomal/Golgi area (Movie 1). BSC1 cells were incubated with 20 nM nontoxic mutant CT(E112D) labeled with Alexa594 (red) at 37°C for 45 min. (B) CT enters BSC1 cells via both vesicles and tubules (arrowheads). BSC1 cells were incubated with 20 nM CT(E112D) at 4°C for 30 min, shifted to 37°C for 2 min, and then the time course of toxin internalization was recorded (inset and Movie 3). Nucleus was stained with Hoechst (blue). (C) Endocytic tubules containing CT (arrowhead) extend from the cell surface toward a perinuclear endosomal compartment in close contact with the Golgi complex (GalT-EGFP, green). BSC1 cells were incubated as described in B. Selected snapshots of time course of CT internalization are shown. The plot shows the time course of CT appearance in endosomes (red line) within the indicated area (red stippled circle); after this brief uptake period, almost no CT reaches the Golgi, including the regions in proximity with the tubules (* in image and green line in plot). Colocalization of CT with the Golgi region was calculated by r analysis as explained in MATERIALS AND METHODS. (D) CT is first transported to endosomes before reaching the Golgi complex. CT(E112D) was added to BSC1/GalT-EGFP cells at 37°C during recording of time lapse. Selected snapshots are shown after 0, 15, and 30 min of CT addition. Colocalization of CT with the Golgi region (stippled box) was calculated as described in C. The plot shows the continuous accumulation of CT in endosomes (red line) and Golgi complex (green line). Bars, 20 μm (whole cell) and 10 μm (cropped areas).

Article Snippet: BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Clinical Proteomics, Membrane, Incubation, Mutagenesis, Labeling, Staining

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Cholera toxin colocalizes with markers of the clathrin-, caveolin-, and Arf6-dependent endocytic pathways. BSC1 cells were incubated with 20 nM CT(E112D) and 50 μg/ml Alexa 647-hTf for 15 min (top two rows) or 45 min at 37°C and fixed. Colocalization between CT and AP-2, EEA1, Arf6, or MHC-I was established by immunofluorescence. Caveolin 1 and EHD1 were tagged with EGFP and transiently expressed in BSC1 cells. Arrow-heads indicate colocalization of CT with AP-2 (presumably in clathrin-coated pits and vesicles), with EEA1 (early endosomes) together with internalized Tf. Representative confocal images from ventral (top row) or middle sections of cells (all others) are shown. Selected regions (stippled-line boxes) were cropped, enlarged, and displayed using an inverted monochrome color scale to aid visualization. Bar, 20 μm.

Article Snippet: BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Incubation, Immunofluorescence

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Clathrin and caveolin-dependent pathways mediate CT internalization. (A) BSC1 cells stably expressing EYFP-LCA cells were incubated with 20 nM CT(E112D) for 30 min at 37°C, washed, and processed for time-lapse recording. A clathrin-coated pit-like structure, selected from the dorsal cell surface (image 1, stippled-line box, and enlarged in images 2 and 3), containing CT was analyzed with the aid of a kymograph plot for clathrin and toxin content over time (images 4–6). (B) CT(E112D) was added during the time-lapse acquisition to BSC1 cells transiently expressing Cav1-EGFP. The time-lapse series (stippled-line box) shows entry of CT into a caveolin 1-containing tubulo-vesicular structure (Movie 7, a and b). The initial short tubular invagination stretches and eventually disconnects from the cell surface. Bars, 20 μm (whole cell) and 5 and 3 μm (cropped regions).

Article Snippet: BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Stable Transfection, Expressing, Incubation

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Combined inhibition of the clathrin-, caveolin-, and Arf6-dependent pathways strongly inhibits CT entry. (A) Inhibition of clathrin-, caveolin-, or Arf6-dependent pathways has minimal effects on CT entry. Representative images of BSC1 cells transiently expressing EGFP-Eps15Δ95-295, Cav1S80E-myc, Arf6Q67L-HA, Arf6T27N-HA for 24–48 h or treated with 100 nM PMA for 30 min (treatment known to disrupt caveolae) are shown. The block in Tf uptake assessed the inhibition of clathrin-mediated endocytosis; inhibition of caveolin-mediated uptake resulted in a 25% reduction in simian virus 40 infectivity (our unpublished data); and interference with the Arf6-dependent pathway by overexpression of Arf6Q67L resulted in the expected vacuolization of the endosomal compartment (Naslavsky et al., 2003 ). (B) Combined inhibition of clathrin- and caveolin-dependent pathways partially inhibits CT entry. Representative images of BSC1 cells treated with 10 mM methyl-β-cyclodextrin for 30 min or overexpressing Dyn1K44A-HA for 48 h are shown. Under both conditions, Tf uptake was strongly inhibited. Although the retention of CT at the cell surface increased upon cholesterol depletion, and in less degree by interference with dynamin-dependent pathways, its internalization was partially prevented. The number of tubules containing CT increased upon expression of dynamin 1 or 2 K44A mutants, which now contain Tf, Cav1-EGFP, Arf6, EGFP-EHD1, and EGFP-MHC-I (arrowheads and Fig. s2 in Supplement). (C) Coexpression of Dyn2K44A and Arf6Q67L or Arf6T27N strongly prevents CT entry and abolished tubule formation. (A–C) Representative confocal images from middle sections of cells (n = 40–60) displayed using an inverted monochrome scale to aid visualization. Transfected cells were identified by imaging EGFP or by staining for the myc or HA epitopes fused to the corresponding overexpressed proteins. In coexpression of Arf6-HA mutants with DynK44A, we used Dyn2K44A-EGFP. Bars, 20 μm.

Article Snippet: BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Inhibition, Expressing, Blocking Assay, Virus, Infection, Over Expression, Transfection, Imaging, Staining

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Decrease of cholesterol or interference with dynamin function inhibits CT transport to the TGN and the ER. Transport of CT to the TGN (A) and ER (B) in cells subjected to different conditions that perturb endocytosis was assessed by colocalization of the toxin with TGN46 or PDI, respectively. BSC1 cells either expressing the indicated dominant proteins for 24–48 h or treated with nocodazole or methyl-β-cyclodextrin for 30 min were incubated with CT(E112D) for 45 min (A) or 90 min (B) at 37°C. Transfected cells were identified as described in Figure 4. For each condition, representative confocal images are shown (out of at least 50 cells analyzed). Selected regions (stippled-line boxes) of the TGN and ER nuclear envelope were cropped, enlarged, and displayed using an inverted monochrome color scale to facilitate inspection of colocalization. Expression of Dyn1K44A or depletion of cholesterol (by treatment with 10 mM methyl-β-cyclodextrin) significantly reduced CT transport to the TGN and ER, whereas inhibition of single endocytic pathways had no effect. In contrast, microtubule depolymerization (by treatment with 10 μM nocodazole) only inhibited retrograde transport the ER. Bars, 20 μm.

Article Snippet: BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Expressing, Incubation, Transfection, Inhibition

Journal: American Journal of Physiology - Renal Physiology

Article Title: Differences in renal BMAL1 contribution to Na + homeostasis and blood pressure control in male and female mice

doi: 10.1152/ajprenal.00014.2020

Figure Lengend Snippet: Antibody information

Article Snippet: Antibody information is shown in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Target Manufacturer Catalog Number Reference Description Dilution Used RRID AQP2 Stressmarq SPC-503D 35 Rabbit, polyclonal IHC: 1:400 AB_10965071 BMAL1 Cell Signaling D2L7G 18 Rabbit, polyclonal IHC: 1:3,000; Western blot analysis: 1:1,000 AB_2728705 NKCC2 Stressmarq SPC-401D 19 Rabbit, polyclonal IHC: 1:10,000 AB_10640877 T53 p-NCC Previously characterized 30 Rabbit, polyclonal IHC: 1:1,000,000 Not applicable Open in a separate window Antibodies were used in this study as previously described ( 18 , 19 , 30 , 35 ).

Techniques: Western Blot

Journal: Pflugers Archiv

Article Title: Characterization of ROMK cellular heterogeneity along the mouse kidney thick ascending limb

doi: 10.1007/s00424-025-03086-4

Figure Lengend Snippet: Imaging pipeline. Cryosections of mouse kidneys were placed on glass slides (top panel) and then processed for RNAscope detection of various mRNAs, combined with immunofluorescent detection of ROMK (lower left panel), for 4i multiplexing immunodetection of several antigens, including ROMK (lower middle panel), or for automated image analysis (lower right panel). For the latter, immunofluorescent stainings for ROMK, NKCC2, and NOS1 were converted to binaries using supervised machine learning (Ilastik) and then analyzed for automated quantification of the cell surface abundance of ROMK

Article Snippet: C , D Close-ups showing TALs stained with our previously published antibodies against NKCC2 and ROMK (Table ); insert in D immunodetection of ROMK with the commercially available antibody from Alomone (Table ); arrowheads – apical localization of ROMK; arrows – perinuclear localization of ROMK Fig. 3 Co-detection of ROMK protein and Kcnj1 mRNA.

Techniques: Imaging, RNAscope, Multiplexing, Immunodetection

Journal: Pflugers Archiv

Article Title: Characterization of ROMK cellular heterogeneity along the mouse kidney thick ascending limb

doi: 10.1007/s00424-025-03086-4

Figure Lengend Snippet: Quantification of ROMK immunostainings. A Percentage of the ROMK-positive apical area in NKCC2-positive TALs in the renal cortex, OMOS, OMIS, and in the macula densa (MD). B Intensity of ROMK immunofluorescence in the cortical TAL and the macula densa (MD) in arbitrary units. Data are presented as means ± standard error of mean; each data point per bar represents a different mouse

Article Snippet: C , D Close-ups showing TALs stained with our previously published antibodies against NKCC2 and ROMK (Table ); insert in D immunodetection of ROMK with the commercially available antibody from Alomone (Table ); arrowheads – apical localization of ROMK; arrows – perinuclear localization of ROMK Fig. 3 Co-detection of ROMK protein and Kcnj1 mRNA.

Techniques: Immunofluorescence

Journal: Pflugers Archiv

Article Title: Characterization of ROMK cellular heterogeneity along the mouse kidney thick ascending limb

doi: 10.1007/s00424-025-03086-4

Figure Lengend Snippet: Immunodetection of ROMK protein at the macula densa. A Immunohistochemical stainings with the commercially available ROMK antibody (Table ); B Immunohistochemical co-staining for NKCC2 (magenta), ROMK (yellow), NOS1 (green) using our previously published ROMK antibody (Table ); arrowheads mark the region of the macula densa (MD); G – glomerulus; scale bar = 50 μm

Article Snippet: C , D Close-ups showing TALs stained with our previously published antibodies against NKCC2 and ROMK (Table ); insert in D immunodetection of ROMK with the commercially available antibody from Alomone (Table ); arrowheads – apical localization of ROMK; arrows – perinuclear localization of ROMK Fig. 3 Co-detection of ROMK protein and Kcnj1 mRNA.

Techniques: Immunodetection, Immunohistochemical staining, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: The β3‐AR agonist BRL37344 ameliorates the main symptoms of X‐linked nephrogenic diabetes insipidus in the mouse model of the disease

doi: 10.1111/jcmm.18301

Figure Lengend Snippet: Multiple repeated BRL37344 1 mg/kg i.p. injections promoted NKCC2 activation in the thick ascending limb of X‐NDI mice. (A) At the end of the 6 i.p injections/24 h experiment, a quantitative reverse transcription polymerase chain reaction was performed on kidneys from X‐NDI treated with saline (CTR, n = 4) or with BRL37344 (BRL, n = 8). Relative quantification of gene expression (RQ) was performed setting the amount of NKCC2 mRNA in CTR as 1. No differences were seen in NKCC2 transcription between the two groups. The experiment was repeated three times and comparable results were obtained. In the scatter plot data were given as mean ± SEM and each dot represents the average of data from three experiments for each mouse. (B) Western blotting with anti‐NKCC2 and antiphosphorylated NKCC2 antibodies was carried out using homogenates prepared from whole kidneys of CTR ( n = 3) and BRL ( n = 9) mice. Representative lanes were reported in the figure. The expression levels of each protein were normalized to total protein content using Stain‐free™ gels technologies. Densitometric analysis showed a two‐fold increase in pNKCC2 (active form), normalized to total NKCC2, in BRL compared to CTR mice. No differences were seen in NKCC2 abundance. The experiment was repeated three times and comparable results were obtained. In the scatter plot, data were given as mean ± SEM and each dot represents the average of data from three experiments for each mouse. **** p < 0.0001 with two‐tailed unpaired Student's t‐test (C) Kidneys from CTR and BRL mice were subjected to immunofluorescence localization of NKCC2 (in green) and pNKCC2 (in green) and counterstained with Evans blue (red) (CTR = 3, BRL = 5). The number of NKCC2‐positive TAL cells and the localization of NKCC2 were similar in two groups but the fluorescence intensity of pNKCC2 was increased in BRL mice. Representative images were shown. The experiment was repeated three times and comparable results were obtained. (bar = 25 μm).

Article Snippet: For quantification of NKCC2, NCC and AQP2 mRNA, real‐time PCR was performed in triplicate using the Applied Biosystem StepONE Real‐time PCR system and the following TaqMan GenExpression Assay (Thermo Fisher Scientific): Mm00490213_m1 for mouse NCC, Mm01275821_m1 for mouse NKCC2 and Mm00437575_m1 for mouse AQP2.

Techniques: Activation Assay, Reverse Transcription, Polymerase Chain Reaction, Saline, Quantitative Proteomics, Gene Expression, Western Blot, Expressing, Staining, Two Tailed Test, Immunofluorescence, Fluorescence

Journal: Physiological Reports

Article Title: Immunofluorescence laser micro-dissection of specific nephron segments in the mouse kidney allows targeted downstream proteomic analysis

doi: 10.14814/phy2.12306

Figure Lengend Snippet: Immunofluorescence laser micro-dissection (IF-LMD) of glomeruli, S1–S2, and S3 proximal tubules. Representative images of IF-LMD of glomeruli, S1–S2 and S3 segments under 40× objective magnification are shown in panels A–B, E–F, and I–J, respectively. Staining was performed using DAPI/Phalloidin. C, G, and K are low magnification (5× objective) bright field microscopy images of kidney sections after dissection of glomeruli, S1–S2 and S3 segments, respectively. Real-time PCR for specific markers (Podocin for glomeruli, SGLT2 for S1–S2, rBAT for S3 segments) was performed on each dissected segment, respectively, as shown in D, H, L, and compared to total kidney used as reference. As expected, each nephron segment had a high level of its corresponding marker as compared to total kidney (* P < 0.05). The purity of RNA sample was demonstrated for S3 segments by the absence of NKCC2, a marker for neighboring thick ascending limb cells in the outer stripe of the outer medulla.

Article Snippet: We used the following Taqman gene expression assays all from Applied Biosystems: SGLT2 (Mm00453831_m1), NKCC2 (Mm00441424_m1), rBAT (Mm00486218_m1), Podocin (Mm01292252-m1) and GAPDH (Mm99999915_g1) as endogenous control .

Techniques: Immunofluorescence, Dissection, Staining, Microscopy, Real-time Polymerase Chain Reaction, Marker

Journal: Physiological Reports

Article Title: Immunofluorescence laser micro-dissection of specific nephron segments in the mouse kidney allows targeted downstream proteomic analysis

doi: 10.14814/phy2.12306

Figure Lengend Snippet: IF-LMF of TAL segments in the outer medulla. Representative images of IF-LMD of TAL segments from the outer medulla in a kidney section stained with Na + -K + -ATPase/DAPI/Phalloidin is depicted in A and B. Real-time PCR on RNA extracted from TAL cells demonstrates the increased expression of NKCC2 (specific marker) in TAL segments as compared to total kidney (* P < 0.05). The purity of the isolated RNA from TAL was demonstrated by the absence of rBAT (marker for neighboring S3 cells in outer stripe).

Article Snippet: We used the following Taqman gene expression assays all from Applied Biosystems: SGLT2 (Mm00453831_m1), NKCC2 (Mm00441424_m1), rBAT (Mm00486218_m1), Podocin (Mm01292252-m1) and GAPDH (Mm99999915_g1) as endogenous control .

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing, Marker, Isolation

Journal: Physiological reports

Article Title: ROMK expression remains unaltered in a mouse model of familial hyperkalemic hypertension caused by the CUL3Δ403-459 mutation.

doi: 10.14814/phy2.12850

Figure Lengend Snippet: Figure 5. Immunostaining of ROMK (green) and AQP2 (red) in the cortex and apical immunostaining of ROMK (green) and total NKCC2 (red) in the medulla of CUL3D403-459/+ kidney versus littermate controls CUL3+/+. These are representative pseudocolored average intensity z- projections of immunofluorescent-stained kidney sections (4 per genotype) and four independent immunostaining experiments, showing the distribution of total protein. Colocalization is in orange. Scale bar = 30 lm.

Article Snippet: Primary antibodies were incubated overnight for 20 h at 4°C at the following concentrations diluted in 1% (v/v) donkey serum in 0.05% (v/v) Triton X-100–PBS: 2 lg/mL for ROMK (https://atlasantibodies.com/), 2 lg/mL for total NKCC2 (Division of Signal Transduction Therapy Unit, University of Dundee), and 0.2 lg/mL for Aquaporin2 (AQP2 antibody, Santa Cruz Biotechnology).

Techniques: Immunostaining, Staining

Journal: Cell reports

Article Title: Magnesium and Calcium Homeostasis Depend on KCTD1 Function in the Distal Nephron

doi: 10.1016/j.celrep.2020.108616

Figure Lengend Snippet: (A) Conditional gene-targeting strategy depicted in the schematic. PvalbCre leads to Cre-mediated recombination of floxed alleles specifically in the DCT1, whereas Aqp2Cre mice target CTs/CDs. Six2Cre mice target the entire nephron, except the CDs. KCTD1 is expressed in the distal nephron (TALs, DCTs, CTs, and CDs). DBA (dolichos biflorus agglutinin) lectin marks CTs/CDs, PNA (peanut agglutinin) lectin marks distal nephron epithelium (discontinuous epithelial staining in TALs, DCTs, and CTs/CDs; continuous labeling of PTs), and LTL (lotus tetragonolobus lectin) marks PTs. Slc3a1 is expressed in PTs, NKCC2 and THP in TALs, NCC in DCTs, Pvalb in DCT1s, and Aqp2 in CTs/CDs. EGF is detected in DCTs and TALs. (B) Serum Mg 2+ levels (in mg/dL) in groups of 2- or 10-month-old PvalbCre + KCTD1 fl/fl or PvalbCre + TFAP2B fl/fl mice and their Cre-negative control littermates (WT). (C) Urine Mg 2+ concentrations (in mEq/L) are increased in 2-month-old PvalbCre + KCTD1 fl/fl mice when compared with their controls, despite normal 24-h urine production in the same mice. (D) Tamoxifen-induced inactivation of KCTD1 at 4–6 weeks of age results in hypomagnesemia when assessed at 5–8 months of age (β-actinCreERT2 + KCTD1 fl/fl mice + TAM). Inactivation of KCTD1 in intestines (VilCre + KCTD1 fl/fl mice) or of KCTD1 or AP-2β in CTs/CDs (Aqp2Cre + KCTD1 fl/fl or Aqp2Cre + TFAP2B fl/fl mice) does not affect serum Mg 2+ levels. (E) Kidneys of adult PvalbCre + KCTD1 fl/fl or PvalbCre + TFAP2B fl/fl mice show reduced protein levels of NCC but not of TRPM6; 12-month-old mice. β-actin as the loading control. (F) Semiquantitative RT-PCR for Trpm6 and Slc12a3 in whole kidneys of 2-month-old Six2Cre + KCTD1 fl/fl mice and their littermate controls. (G) Heatmaps show expression levels of genes implicated in the regulation of Mg 2+ homeostasis in the kidney. RNA-seq data from kidneys of mice with induced inactivation of AP-2β in the adult (4-month-old β-actinCreERT2 + TFAP2B fl/fl mice treated with TAM at 6 weeks of age) and their Cre controls (left) or from kidneys of P8 Six2Cre + KCTD1 fl/fl mice and their Cre control littermates . Differentially expressed genes (DEGs) (>1.5-fold change [FC] in expression; false discovery rate [FDR] < 0.05) are indicated. In both mutant groups, the DCT-expressed genes EGF, Slc12a3, and Pvalb are significantly downregulated. Scale shows FC normalized to the control group average. (H) EGF (arrow) is detected in Pvalb + DCT1s at the apical cell membrane (EGF in green, Pvalb in red). No EGF is detected in kidneys of EGF −/− mice. Scale bar, 50 µm. DAPI stains nuclei blue. Mg 2+ serum levels are not significantly reduced in EGF −/− mice compared with their WT littermates at 2 or 4 months of age. Graphs represent data as means ± SEM. Semiquantitative RT-PCRs performed in triplicate. p values are shown (two-tailed, unpaired t test). Related to .

Article Snippet: Antibodies against NKCC2 (Cell Signaling Technology, Cat# 38436, RRID:AB_2799134), NCC (Millipore Cat# AB3553, RRID:AB_571116) and TRPM6 (Thermo Fisher Scientific Cat#PA5–77326, RRID:AB_2736741) were used as well.

Techniques: Staining, Labeling, Negative Control, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Sequencing, Mutagenesis, Membrane, Two Tailed Test

Journal: Cell reports

Article Title: Magnesium and Calcium Homeostasis Depend on KCTD1 Function in the Distal Nephron

doi: 10.1016/j.celrep.2020.108616

Figure Lengend Snippet: (A) KCTD1 is expressed in TALs (arrows): b-galactosidase (red) in KCTD1 WT/lacZ mice is observed in NKCC2 + TALs (green); 12-month-old KCTD1 WT/lacZ mouse. Scale bar, 50 µm. (B) THP (green, arrows) marks TALs, which are dilated in aged KCTD1 −/− mice (8-month-old). Scale bar, 50 µm. (C) Top: mild dilatation of NKCC2 + TALs (green, white arrows) is observed in 1-month-old KCTD1 −/− mice. Scale bars, 100 µm. Bottom: 12-month-old KCTD1 −/− mice, expressing b-galactosidase (red) from the endogenous KCTD1 locus, show extensive TAL dilatations with discontinuous and irregular expression of NKCC2 (green; yellow arrow); 12-month-old KCTD1 −/WT mice show normal NKCC2 labeling in normal-appearing β-gal + TALs (white arrow). Scale bars, 50 µm. (D) Western blot with kidney lysates for NKCC2 and NCC. Kidneys of Six2Cre + KCTD1 fl/fl mice at birth (P0) show no major reduction in NKCC2 and NCC protein levels. In contrast, 2-month-old Six2Cre + KCTD1 fl/fl or KCTD1 −/− mice show a strong decrease in NKCC2 and NCC protein levels compared with their control littermates. β-actin as loading control. (E) WT mice increase their 24-h urine production when treated with the NKCC2 inhibitor furosemide. Diminished levels of NKCC2 in KCTD1 −/− mice correlate with a lack of a response to furosemide; 2-month-old mice (n = 4). (F) Few apoptotic cells (TUNEL + , arrow) in dilated distal nephron tubules in 9-month-old KCTD1 −/− mice are being extruded into their lumina. No apoptotic cells in 9-month-old WT littermates. Scale bar, 50 µm. (G) AQP2 is localized at the apical luminal membrane in β-gal + -expressing CTs/CDs of KCTD1 −/− mice even without administration of desmopressin (dDAVP; with similar effects like vasopressin in CTs/CDs), whereas AQP2 is localized in the cytoplasm in WT littermates without dDAVP administration and becomes only apically localized when treated with dDAVP; 4-month-old mice are shown. Scale bars, 20 µm. DAPI stains nuclei blue.

Article Snippet: Antibodies against NKCC2 (Cell Signaling Technology, Cat# 38436, RRID:AB_2799134), NCC (Millipore Cat# AB3553, RRID:AB_571116) and TRPM6 (Thermo Fisher Scientific Cat#PA5–77326, RRID:AB_2736741) were used as well.

Techniques: Expressing, Labeling, Western Blot, Control, TUNEL Assay, Membrane

Journal: Cell reports

Article Title: Magnesium and Calcium Homeostasis Depend on KCTD1 Function in the Distal Nephron

doi: 10.1016/j.celrep.2020.108616

Figure Lengend Snippet: (A) Semiquantitative RT-PCR in kidneys of 2-month-old KCTD1 −/− and Six2Cre + KCTD1 fl/fl mice shows a significant decrease in NKCC2 (encoded by Slc12a1) transcripts compared with their littermate controls. P3 Six2Cre + KCTD1 fl/fl mice show normal NKCC2 levels, and P8 Six2Cre + KCTD1 fl/fl mice show a modest reduction in NKCC2 expression. Inducible inactivation of KCTD1 with TAM at P9 in b-actinCreERT2 + KCTD1 fl/fl mice results in strong reduction of NKCC2 expression in the kidney. Claudin-16 and claudin-19 expression is strongly reduced in kidneys of 2-month-old KCTD1 −/− and Six2Cre + KCTD1 fl/fl mice, whereas claudin-14 expression is increased. Expression of ATP1a1 or PTH1R is decreased in kidneys of 2-month-old KCTD1 −/− and Six2Cre + KCTD1 fl/fl mice but not at P3 before nephron maturation. Values normalized to their controls. (B) The expression of CLC-NKB is increased in kidneys of 2-month-old KCTD1 −/− and Six2Cre + KCTD1 fl/fl mice. This is not observed before the manifestation of the distal nephron maturation defect (normal at P8). Values are normalized to their controls. (C) Total expression levels of claudin-16 and claudin-19 in kidneys of P3, P8, and 2-month-old Six2Cre + KCTD1 fl/fl mice and their littermate controls. (D) Semiquantitative RT-PCR analysis in kidneys of 2-month-old KCTD1 −/− and Six2Cre + KCTD1 fl/fl mice shows a significant decrease in Slc34a1 and Slc34a3 transcripts. Values normalized to their controls. (E) Semiquantitative RT-PCR analysis in kidneys of 2-month-old KCTD1 −/− and Six2Cre + KCTD1 fl/fl mice shows a significantly increased expression of the Ca 2+ channels TRPV5 and TRPV6. NCX1 and calbindin-D 28K expression is not reduced in kidneys lacking KCTD1. Values normalized to their controls. (F) Summary of expression changes of key regulators of urinary Ca 2+ reabsorption shows similar results when comparing kidneys of 2-month-old KCTD1 −/− and Six2Cre + KCTD1 fl/fl mice. (G) Immunolabeling of kidneys from 6-month-old Six2Cre + KCTD1 fl/fl mice and their WT controls for active β-catenin, claudin-14, claudin-19, and TRPV5 shows localization of these proteins in dilated distal tubules of Six2Cre + KCTD1 fl/fl mice. DAPI stains nuclei blue. Scale bars, 20 µm Graphs represent data as means ± SEM. Semiquantitative RT-PCRs performed in triplicate with n > 6 samples/group. p values are shown (two-tailed, unpaired t test).

Article Snippet: Antibodies against NKCC2 (Cell Signaling Technology, Cat# 38436, RRID:AB_2799134), NCC (Millipore Cat# AB3553, RRID:AB_571116) and TRPM6 (Thermo Fisher Scientific Cat#PA5–77326, RRID:AB_2736741) were used as well.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunolabeling, Two Tailed Test

Journal: Cell reports

Article Title: Magnesium and Calcium Homeostasis Depend on KCTD1 Function in the Distal Nephron

doi: 10.1016/j.celrep.2020.108616

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies against NKCC2 (Cell Signaling Technology, Cat# 38436, RRID:AB_2799134), NCC (Millipore Cat# AB3553, RRID:AB_571116) and TRPM6 (Thermo Fisher Scientific Cat#PA5–77326, RRID:AB_2736741) were used as well.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Pflugers Archiv

Article Title: A novel mouse model for familial hypocalciuric hypercalcemia (FHH1) reveals PTH-dependent and independent CaSR defects

doi: 10.1007/s00424-024-02927-y

Figure Lengend Snippet: Expression of key proteins involved in renal calcium, magnesium, and salt handling. Protein abundance was assessed by immunoblotting in protein homogenates from kidneys from wildtype (WT/WT), Pth KO/ Casr BCH002 , WT/ Casr BCH002 and Pth KO/ Casr BCH002 mice. Protein expression of ( A , B ) NKCC2 (120 kDa), ( C , D ) NCC (130 kDa), ( E , F ) TRPV5 (75 kDa) ( G , H ) Calbindin D28k (28 kDa), and ( I , J ) TRPM6 (230 kDa) were normalized to the corresponding ponceau S staining. Values are presented as means ± SD together with single values ( n = 6/group). Data were analysed with 2-way ANOVA with Tukey correction for multiple comparisons between PTH and BCH genotypes. * p < 0.05

Article Snippet: Antibodies used were directed against NaPi-IIa (1:2000) [ ], NaPi-IIc (1:1000) [ ], total NCC (1:2000; a kind gift of J. Loffing [ ]), NKCC2 (1:2000, a kind gift of J. Loffing) [ ], NHE3 (1:1000; StressMarq; SPC-400D), TRPV5 (1: 500, a kind gift by O. Bonny, [ ]), TRPM6 (1:2000, a kind gift by J. Loffing, [ ]), the vitamin D receptor (Vdr) (1:500 Santa Cruz), Cyp24a1 (1:1000, Proteintech), the Ca 2+ -sensing receptor (CaSR) (1:500, Thermo Fisher) and calbindin D28k (1:500, Swant).

Techniques: Expressing, Western Blot, Staining

Journal: International journal of molecular sciences

Article Title: Sex Modulates Response to Renal-Tubule-Targeted Insulin Receptor Deletion in Mice.

doi: 10.3390/ijms24098056

Figure Lengend Snippet: Figure 4. Western blotting of key sodium- and water-reabsorptive routes in kidney regional homogenates—(A) representative Western blots of cortex, outer medullary, or inner medullary homogenates prepared from male (M) and female (F) WT and KO mice probed with specific antibod- ies against the three ENaC subunits, NKCC2, NCC, and aquaporin 2 (AQP2); (B) summary of band densities (mean ± sem) normalized by Ponceau Red stain for all Western blots (n = 6/genotype/sex); letters above bars indicate results of Tukey’s multiple comparisons test (only conducted when a main factor < 0.05), with “A” assigned to the highest mean and all means not different from it, followed by “B”, etc. Bars with letters “not in common” are significantly different from each other, e.g., “AB” versus “C”; (C) summary of two-way ANOVA results (p < 0.05 considered significant, in bold); CTX—cortex; OM—outer medulla; IM—inner medulla.

Article Snippet: Primary antibodies used for Western blotting were as follows: (1) InsR-β (A303–712A, polyclonal rabbit, Bethyl Laboratories, Montgomery, TX, USA); (2–4) α-, β-, and γ-ENaC (our own rabbit polyclonals) [18]; (5–7) NKCC2, AQP2, and NCC (our own rabbit polyclonals) [12]; (8) p-126-serine NKCC2 (rabbit polyclonal, a kind gift from Mark Knepper); (9) PEPCK (sc271019, rabbit polyclonal, Santa Cruz Biotechnology, Dallas, TX, USA); (10) FBP1 (109020, monoclonal rabbit, Abcam, Waltham, MA, USA); (11) G6PC (PAS-42541, polyclonal rabbit, Invitrogen, Waltham, MA, USA); (12) SGLT1 (NBP2-20338, polyclonal rabbit, NovusBio, Littleton, CO, USA); (13) SGLT2 (ab37296, polyclonal rabbit, Abcam), and (14) SNAT3 (14315, polyclonal rabbit, Proteintech, San Diego, CA, USA).

Techniques: Western Blot, Staining