Immunolocalization of Protein Kinase C Isoenzymes α, βI and βII in Rat Kidney

Western Blot Analysis

After anesthesia and opening of the abdominal cavity, kidneys were rapidly excised and dissected on ice in cortical and medullary parts. Pieces were immediately immersed into liquid nitrogen. Total cellular proteins were obtained by pulverizing the tissue and dissolving the powder in lysis buffer (20 mM Hepes, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 4 mM ethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetra-acetic acid, 4 mM ethylenediaminetetra-acetic acid, 1mM phenylmethylsulfonyl fluoride, 30 μg/ml aprotinin, 5 μg/ml leupeptin, 10 μg/ml trypsin inhibitor, and 1 mM dithiothreitol). Homogenization was followed by centrifugation (17,000 × g, 15 min, 4°C). Protein content was determined as described by Bradford (17), using a commercial protein assay (Bio-Rad, Munich, Germany) and bovine serum albumin (Serva, Heidelberg, Germany) as a standard. Samples were diluted 1:4 with Roti®-Load sample buffer (Roth, Karlsruhe, Germany) and boiled for 2 min at 95°C. The following steps were performed at room temperature. Samples of 10 μg of protein (cortex) or 20 μg of protein (medulla), respectively, were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 8% acrylamide gels in a Mini-PROTEAN II Electrophoresis Cell (Bio-Rad). For determination of molecular weight, a 10-kD protein ladder (Life Technologies, Eggenstein, Germany) was used. After gel electrophoresis (60 mA/gel, 70 min), proteins were transferred to nitrocellulose paper of 0.45-μm pore size (Schleicher & Schuell, Dassel, Germany). Membranes were blocked for 90 min with phosphate-buffered saline (PBS) containing 2% gelatin, 2% normal goat serum, 2% low-fat milk powder, and 0.02% Tween 20 and rinsed twice with PBS containing 0.02% Tween 20 (PBST). Thereafter, the PKC antibodies (anti-PKC α at 0.3 μg/ml; anti-PKC βI and anti-PKC βII at 0.5 μg/ml) were incubated for 30 min in PBS containing 10 mM ethylenediaminetetra-acetic acid, 2% normal goat serum, and 0.2% gelatin. Blots were rinsed twice with PBST and washed twice for 15 min with PBST. The secondary horseradish peroxidase-conjugated antibody was incubated at a concentration of 0.1 μg/ml for 30 min in PBST containing 0.2% gelatin. Blots were rinsed twice with PBST and washed three times for 15 min with PBST. The immunoreactive bands were visualized by enhanced chemiluminescence system onto a Hyperfilm-ECL™ (Amersham, Buckinghamshire, United Kingdom). The specificity of the PKC antibody binding was confirmed by peptide neutralization of the antibodies with their corresponding peptide. For neutralization, antibodies were incubated with a 10-fold (by weight) excess of peptide antigen overnight at 4°C before incubating with the blotting membrane.

Immunohistochemistry

After anesthesia, the left cardiac ventricle was exposed through subcostal incision of the abdominal cavity and subsequent opening of the pericardium. The tip of a perfusion system was placed into the left ventricle, and the arterial system was perfused for 1 min with PBS (120 mM NaCl, 16 mM Na₂HPO₄, 2,9 mM KH₂PO₄) to clear the kidneys of blood and subsequently for 10 min with 150 ml of a fixation solution (4% paraformaldehyde and 3% sucrose in phosphate buffer). Both solutions were at room temperature. Kidneys were removed, cut into slices (3 mm in thickness) displaying cortex and outer and inner medulla, and incubated for 1 h in the fixation solution at 4°C. After rinsing four times in PBS for 15 min, kidney slices were dehydrated in 30% sucrose in PBS for 12 h at 4°C. Thereafter, kidney slices were frozen in isopentane precooled by liquid nitrogen and stored at -80°C until further use. Cryosections of approximately 16 μm were made at -20°C and transferred onto gelatin-coated glass slides. The following steps were performed at room temperature. After preincubation for 45 min in PBS containing 4% normal goat serum, 1% bovine serum albumin, and 0.25% Triton X-100 in PBS, sections were incubated for 4 h with the respective anti-PKC antibody (anti-PKC α at a final concentration of 1.0 μg/ml; anti-PKC βI and anti-PKC βII at 2.0 μg/ml). For negative control, the primary PKC antibodies were preabsorbed with their corresponding peptides by incubating them with a 10-fold excess of peptide antigen in PBS for 2 h at room temperature before incubating the antibody-peptide solution with the sections. After washing the sections three times for 5 min with PBS, they were incubated for 1.5 h with Texas Red-conjugated goat anti-rabbit IgG (final concentration, 5 μg/ml). Sections were then washed twice for 10 min in PBS and mounted in FluorSave™ (Calbiochem, San Diego, CA) as fading retardant.

Double staining experiments were performed with each one of the PKC antibodies and a primary antibody against the respective tissue epitope. To study the expression of PKC in glomerular mesangial cells, a monoclonal antibody (mAb) against Thy-1 antigen (final concentration, 2 μg/ml) was applied. For staining of glomerular endothelial cells, an mAb against von Willebrand factor (final concentration, 10 μg/ml) was used (18), whereas the thick ascending limb (TAL) was labeled by a polyclonal antibody (goat antiserum) against Tamm-Horsfall protein (19) (final concentration, 250 μg total protein ml). To further identify the anti-PKC βII-stained cortical interstitial cells, double labeling was performed with a mAb against ecto-5′-nucleotidase (1:100), which is expressed in cortical fibroblasts (20), or a mAb against OX6 (final concentration, 5 μg/ml), which is an MHC II gene product expressed on the surface of antigen-presenting cells (21) such as cortical and medullary interstitial dendritic cells (22). The following secondary antibodies were used: goat anti-rabbit IgG conjugated with carbocyanine (Cy2) for PKC antibodies (except for double staining with Tamm-Horsfall protein, see below); goat anti-mouse IgG conjugated with indodicarbocyanine (Cy5) for antibodies against Thy-1, von-Willebrand-factor, OX6, and ecto-5′-nucleotidase. For the antibody against Tamm-Horsfall protein, mouse anti-goat IgG conjugated with Cy2 was used. In this case, donkey anti-rabbit IgG conjugated with Cy5 was used as secondary antibody to the respective PKC antibodies. The final concentration of all secondary antibodies was 5 μg/ml.

For labeling of filamentous actin in foot processes of podocytes or proximal tubular brush border, rhodamine-conjugated phalloidin (dilution 1:20) was used, whereas rhodamine-conjugated peanut agglutinin (PNA), which has been shown to stain a subpopulation of cells in CCD of the rabbit (including type B cells) (23), was used to label cells in the CCD (final concentration, 5 μg/ml).

Sections were studied by confocal laser scanning microscopy (Zeiss LSM 410 Invert; Jena, Germany). For immunohistochemical reference, cryosections of rat brain were used, which showed specific immunostaining with all three PKC isoenzyme antibodies.

Antibodies and Staining Substances

For Western blot and immunohistochemistry analysis, polyclonal rabbit antibodies against the following epitopes were used: PKC α (human amino acid residues 651 to 672), PKC βI (human amino acid residues 656 to 671), PKC βII (human amino acid residues 657 to 673). These epitopes correspond to the last amino acids at the carboxy-terminal catalytic domain in the V5 region of the PKC. The antibodies and their corresponding peptides as well as anti-rabbit-IgG-horseradish peroxidase secondary antibody were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). mAb against Thy-1 was purchased from Seikagaku Corp. (Tokyo, Japan), mAb against von Willebrand factor from Boehringer Mannheim (Mannheim, Germany), and goat antiserum against Tamm-Horsfall protein from ICN (Eschwege, Germany). mAb against MHC class II (OX6) was purchased from Harlan Sera-Lab Ltd. (Loughborough, United Kingdom). mAb against ecto-5′-nucleotidase was a generous gift from B. Kaissling (Anatomisches Institut der Universität, Zürich, Switzerland). Rhodamine-conjugated phalloidin was obtained from Molecular Probes (MoBiTec, Göttingen, Germany), and rhodamine-conjugated PNA was from Vector Laboratories (Burlingame, CA). Texas Red- and Cy2-conjugated goat anti-rabbit IgG as well as Cy5-conjugated goat anti-mouse IgG, Cy5-conjugated mouse anti-goat IgG, and Cy5-conjugated donkey anti-rabbit IgG were obtained from Dianova (Hamburg, Germany).

Western Blot Analysis

In both cortex and medulla, PKC α and PKC βI stained primarily at approximately 80 kD, whereas PKC βII was detected at approximately 83 kD (Figure 1). These molecular weights are in agreement with the literature (24,25,26,27). The smaller proteins detected by antibodies against PKC α and βI were inhibited by antibody preabsorption with the respective peptide and could represent proteolytic fragments due to enzymatic degradation.

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Figure 1.

(A) Western blot analysis showing the expression of protein kinase C (PKC) α, βI, and βII in rat renal cortex and medulla at approximately 80 kD. (B) Antibody preabsorption with the respective peptide inhibited all bands on the Western blots completely.

Immunohistochemistry

The distribution of PKC isoenzymes α, βI, and βII in rat kidney is depicted in Table 1 and illustrated in Figures 2 3,4,5,6,7,8. The specificity of all three PKC isoenzyme polyclonal antibodies was tested by preimmunoadsorption with the corresponding peptides. This treatment consistently abolished specific binding of the fluorescence secondary antibody.

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Figure 2.

Immunolocalization of PKC α, βI, and βII in rat glomerular podocytes. Double staining with antibodies against PKC α, PKC βI, and PKC βII, respectively (red fluorescence), and rhodamine-conjugated phalloidin, which labels filamentous actin in foot processes of podocytes and proximal tubular brush border (green fluorescence). Scale bars, 25 μm. (A) PKC α. Bodies of podocytes were stained (arrowheads). (B) PKC βI. In podocytes, a faint signal for PKC βI may be present. A clear-cut positive labeling, however, could not be assigned unequivocally (arrowhead). (C) PKC βII. No significant signal was detected in podocytes after labeling with PKC βII antibodies.

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Figure 3.

Immunolocalization of PKC α, βI, and βII in rat glomerular mesangial cells. Double staining with antibodies against PKC α, PKC βI, and PKC βII, respectively (red fluorescence), and anti-Thy-1, which labels mesangial cells (green fluorescence) (A through C). Scale bars, 25 μm. PKC α (A) and PKC βI (B) were detected in glomerular mesangial cells (arrowheads). (C) PKC βII labeled round, cell-like structures within the mesangial area, which morphologically appeared not to represent mesangial cells (arrowheads). (D) Colocalization of PKC βII (red fluorescence) and anti-OX6 (green fluorescence), a marker for MHC II-positive, antigen-presenting cells (arrowheads). There was no staining of OX6-positive cells by anti-PKC α or anti-PKC βI (not shown).

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Figure 4.

Immunolocalization of PKC α and βI in rat proximal tubule. Double staining with antibodies against PKC α and PKC βI, respectively (red fluorescence), and rhodamine-conjugated phalloidin (green fluorescence), which stains the brush border of proximal tubule. Scale bars, 25 μm. PKC α (A) and PKC βI (B) in cortical labyrinth. Proximal tubules showed no immunofluorescence in the S1 segments (which were defined by a dense and high brush border and close localization to glomeruli [arrows] or direct connection with the parietal epithelium of the urinary pole of the glomerulus), but a very prominent staining of the brush border of S2 segments (which were defined by a less dense brush border that was reduced in height) with anti-PKC α or anti-PKC βI antibodies (arrowheads). PKC α (C) and PKC βI (D) in medullary rays of the outer stripe of outer medulla. Both PKC α and PKC βI were detected in the brush border of S3 segments (arrowheads).

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Figure 5.

Immunolocalization of PKC α and βI in thin limbs and thick ascending limbs (TAL). Double staining with antibodies against PKC α and PKC βI (red fluorescence), and anti-Tamm-Horsfall protein (green fluorescence), which labels the luminal membrane of TAL. Scale bars, 25 μm. (A) PKC α in cortex. Luminal membrane of cortical TAL showed a positive signal for PKC α (arrowheads). (B) PKC α in inner stripe of outer medulla. PKC α was detected in luminal membrane of medullary TAL (arrowheads). (C) PKC βI in cortex. Like PKC α, PKC βI could be detected in luminal membrane of cortical TAL (arrowheads). (D) PKC βI in inner stripe of outer medulla. Luminal membrane of medullary TAL was immunopositive for PKC βI (arrowheads).

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Figure 6.

Immunolocalization of PKC α and βI in cortical collecting duct (CCD). Double staining with antibodies against PKC α and PKC βI, respectively (red fluorescence), and rhodamine-conjugated peanut agglutinin (PNA; green fluorescence), which has been shown to stain a subpopulation of cells in CCD of the rabbit (including type B cells) (23). As shown, the appearance of the staining with PNA in rat CCD resembles the staining in rabbit CCD as indicated by predominant labeling of a subpopulation of cells that exhibit an intercalated cell-like appearance. Scale bars, 25 μm. (A) PKC α. In CCD, PKC α was expressed in the luminal membrane of both PNA-negative cells (arrowheads) and part of PNA-positive cells (asterisk). A part of PNA-positive cells, however, was PKC α-immunonegative (arrows). (B) PKC βI. Immunofluorescence for PKC βI was detected in the luminal membrane of PNA-positive cells (arrows).

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Figure 7.

Immunolocalization of PKC α, PKC βI, and PKC βII in medullary collecting duct (MCD) (inner medulla). Scale bars, 25 μm. (A) PKC α. The basolateral membrane of MCD was intensely stained (arrows). (B) PKC α. Only MCD were immunopositive (arrows), whereas thin limbs were not stained. (C) PKC βI. Staining of the luminal membranes of both MCD (arrows) and thin limbs (arrowhead). (D) PKC βII. Anti-PKC βII stained predominantly the basolateral membrane of MCD (arrows). In addition, medullary interstitial cells were labeled (asterisk).

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Figure 8.

Further identification of cortical interstitial cells expressing PKC βII. Double staining with antibodies against PKC βII (red fluorescence) and ecto-5′-nucleotidase (green fluorescence), which is a marker for fibroblasts (A and B), or OX6 (green fluorescence), which labels MHC II-positive, antigen-presenting cells (C), respectively. Scale bars, 25 μm. (A) One of few anti-ecto-5′-nucleotidase-positive fibroblasts that were also labeled by anti-PKC βII (arrow). (B) PKC βII in a dendritic-shaped cell (red; arrow) surrounded by PKC βII-negative but anti-ecto-5′-nucleotidase-positive fibroblasts (green; arrowheads). (C) PKC βII in an OX6-positive, dendritic-shaped cell (arrow).

PKC α

Within glomeruli, immunostaining for PKC α included the bodies but not the foot processes of podocytes as well as mesangial cells (Figures 2A and 3A). PKC α could not be detected in glomerular endothelial cells as studied by double labeling with antibodies against von Willebrand factor (not shown). In the cortical labyrinth, there was no signal detected in S1 segments of proximal tubules after staining with anti PKC α (Figure 4A). In contrast, a very bright immunofluorescence was localized to the brush border of S2 segments. S1 segments were identified by a dense and high brush border and close localization to glomeruli or direct connection with the parietal epithelium of the urinary pole of the glomerulus. In comparison, S2 segments were defined by a less dense brush border that is reduced in height (28). Immunostaining of proximal tubules was very bright within the brush border of S3 segments in the outer stripe of outer medulla (Figure 4C). No visible immunofluorescence was detected in thin limbs of Henle's loop, whereas the luminal membranes of cortical and medullary TAL were labeled (Figure 5, A and B). To characterize the expression of PKC α in CCD, PNA was used, which has been shown to stain a subpopulation of cells in CCD of the rabbit (including type B cells) (23). It was observed in the present study that the appearance of the staining with PNA in rat CCD resembles the staining in rabbit CCD as indicated by predominant labeling of a subpopulation of cells that exhibit an intercalated cell-like appearance (Figure 6, A and B). Because the subtype of cells in rat CCD stained by PNA has not yet been established, however, cells of the CCD that are stained by PNA in the present study are referred to as PNA-positive cells. It was found that anti-PKC α stained the luminal membrane of both PNA-negative cells and part of PNA-positive cells in CCD (Figure 6A). In MCD, a bright labeling of the basolateral membranes was observed (Figure 7A).

PKC βI

Anti-PKC βI stained mesangial cells (Figure 3B). In podocytes, a faint signal for PKC βI may have been present. A clear-cut positive labeling, however, could not be assigned unequivocally (Figure 2B). Anti-PKC βI did not label endothelial cells (not shown). As shwon for PKC α, PKC βI immunofluorescence was observed in the S2 and S3 segments of proximal tubular brush border (Figure 4, B and D), whereas no significant signal was detected in S1 segments (Figure 4B). Unlike anti-PKC α, anti-PKC βI labeled the luminal membrane of thin limbs of Henle's loop (Figure 7C). As shown for PKC α, PKC βI was detected at the luminal membrane of cortical and medullary TAL (Figure 5, C and D). Compared with anti-PKC α, anti-PKC βI stained predominantly the luminal membrane of PNA-positive cells in CCD (Figure 6B) and the luminal membrane in MCD (Figure 7C).

PKC βII

In contrast to PKC α and PKC βI, PKC βII could not be detected in glomerular mesangial cells (Figure 3C). In some glomeruli, however, anti-PKC βII stained round cell-like structures that appeared to be localized within the mesangial area and that were double-stained by an antibody against OX6, which is a marker for MHC II-positive, antigen-presenting cells (21) (Figure 3D). Podocytes (Figure 2C) and glomerular endothelial cells (not shown) appeared PKC βII-immunonegative. Furthermore, there was no detectable staining with anti-PKC βII in proximal tubules, TAL, or CCD (not shown). In MCD, anti-PKC βII labeled predominantly the basolateral membrane (Figure 7D). In contrast to PKC α or βI, anti-PKC βII stained both cortical and medullary interstitial cells. A significant portion of both cortical and medullary PKC βII-positive interstitial cells was colabeled by anti-OX6 antibodies (Figure 8C). In comparison, anti-PKC βII-labeled cortical interstitial cells included only a very minor fraction of ecto-5′-nucleotidase-positive fibroblasts (Figure 8, A and B).