Journal: Physiology international

Article Title: The role of TRPC6 calcium channels and P2 purinergic receptors in podocyte mechanical and metabolic sensing

doi: 10.1556/2060.2021.00205

Figure Lengend Snippet: (A) Immunofluorescence localization of TRPC6 (red) with GCaMP3 (G3) co-labeling (green), or P2Y2 (green) in wild type (WT), TRPC6 KO or P2Y2 KO mouse kidney tissue sections as indicated. Overlay of green and/or red channels with the nuclear stain DAPI (blue) is shown. For TRPC6 WT and KO tissues, individual red and green channels are included separately in insets. Note the strong TRPC6 (co-localized with Pod-G3) or P2Y2 labeling in podocytes (arrows) in WT, but the absence of TRPC6 or P2Y2 labeling in TRPC6 KO or P2Y2 KO mouse kidney sections, respectively. (B) Representative intravital MPM images of the overview of the entire renal cortex area that is available for MPM imaging (left, tile-scan) or a single glomerulus with high-magnification (right, single xy frame) from a WT Pod-GCaMP5 (G5, green)/tdTomato (red) mouse. The fibrous renal capsule was visualized with second harmonic generation (SHG, cyan). The plasma was labeled using Albumin-Alexa Fluor 680 (grey) or Albumin-Alexa Fluor 594 (red). Note the high number of renal cortical glomeruli (left panel) available for MPM imaging and the clear view of individual podocytes co-expressing G5 and tdTomato (yellow, right panel). G: glomerulus. Scale bar is 20 μm for panel A and 100 μm for panel B

Article Snippet: Male and female, 4–8 weeks old wild-type (WT), TRPC6 and P2Y2 knockout (KO) C57BL6/J mice (Jackson Laboratory, Bar Harbor, ME) were used in all experiments.

Techniques: Imaging, Immunofluorescence, Labeling, Staining, Clinical Proteomics, Expressing

Journal: Physiology international

Article Title: The role of TRPC6 calcium channels and P2 purinergic receptors in podocyte mechanical and metabolic sensing

doi: 10.1556/2060.2021.00205

Figure Lengend Snippet: (A) Representative single-frame xy image at baseline (left) and maximum projection image (MPI) of a 40 second time-lapse after injury (right) of two glomeruli in WT Pod-GCaMP3 mice. A short pulse of a laser beam focused on a small focal point of the efferent arteriole (EA) endothelium of one glomerulus (G i , denoted by green circle) caused localized endothelial injury and formation of a microthrombus in the lumen of the EA. The other adjacent glomerulus (G c ) was left intact and served as control. Note the glomerular tuft distension indicating capillary pressure elevation, increased podocyte [Ca 2+ ] i (increase in GCaMP3 fluorescent intensity-bright green, including a cell-to-cell propagating podocyte [Ca 2+ ] i wave indicated by a green arrow), and albumin leakage into the Bowman’s space (red around the glomerular capillaries) only in the injured glomerulus (G i )(a time-lapse recording of the same preparation is shown in Supplement Movie 1 ). GCaMP3 images are included (bottom) that illustrate the changes in podocyte [Ca 2+ ] i in response to glomerular capillary pressure elevations over time (baseline at time of injury, and 30–40–50 s later). Note the high [Ca 2+ ] i in distant podocytes appearing simultaneously (P 1 –P 2 ), or with a delay in a cell-to-cell propagating podocyte [Ca 2+ ] i wave (P 1 –P 3 –P 4 ). (B) Summary of podocyte [Ca 2+ ] i responses (normalized to baseline, dashed line) to mechanical overload in WT, TRPC6-KO, and P2Y2-KO animals. Data represent mean ± SEM, ****: P < 0.0001, 2 glomeruli/mouse from n = 5 mice in each group). (C) Representative recordings of single-cell podocyte [Ca 2+ ] i responses over time illustrating the dynamics of [Ca 2+ ] i changes in four different podocytes during capillary pressure elevation (time of microthrombus formation indicated by vertical line). Different colors represent P 1 –P 4 single podocytes matching those shown in panel A (bottom). Time-course of albumin appearing in the Bowman’s space is indicated by red line

Article Snippet: Male and female, 4–8 weeks old wild-type (WT), TRPC6 and P2Y2 knockout (KO) C57BL6/J mice (Jackson Laboratory, Bar Harbor, ME) were used in all experiments.

Techniques: In Vivo, Control, Single Cell

Journal: Physiology international

Article Title: The role of TRPC6 calcium channels and P2 purinergic receptors in podocyte mechanical and metabolic sensing

doi: 10.1556/2060.2021.00205

Figure Lengend Snippet: (A-B) Representative images of a freshly dissected and in vitro microperfused glomerulus (G) from a WT Pod-GCaMP3 mouse kidney at baseline (A) and after obstructing the efferent arteriole (EA) lumen by a micropipette (B, mechanical overload). Note the increased podocyte GCaMP3 fluorescence (green, arrows) after glomerular capillary pressure elevation (B) compared to baseline (A). DIC overlay is shown with GCaMP3 fluorescence. Afferent arteriole (AA) perfusion was kept constant. Bar is 20 μm. The same preparation is shown in Supplement Movie 2 . (C) Summary of the changes in podocyte [Ca 2+ ] i (GCaMP3 fluorescence normalized to baseline (dashed line), F max /F 0 ) in response to mechanical overload in preparations from WT, TRPC6-KO, and P2Y2-KO animals. (D) Changes in glomerular diameter in response to mechanical overload in each group. Data represent mean ± SEM, ns: not significant, ****: P < 0.0001, n = 10 in each group.

Article Snippet: Male and female, 4–8 weeks old wild-type (WT), TRPC6 and P2Y2 knockout (KO) C57BL6/J mice (Jackson Laboratory, Bar Harbor, ME) were used in all experiments.

Techniques: In Vitro, Fluorescence