Identification and localization of TRPC channels in the rat kidney - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:44 |只看该作者 |倒序浏览 |打印

作者：Monu Goel, William G. Sinkins, Cheng-Di Zuo, Mark Estacion, and William P. Schilling,作者单位：2 Rammelkamp Center for Education and Research, MetroHealth Medical Center, and 1 Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio

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      【摘要】
   It is well established that transient receptor potential (TRP) channels are activated following stimulation of G protein-coupled membrane receptors linked to PLC, but their differential expression in various cells of the renal nephron has not been described. In the present study, immunoprecipitations from rat kidney lysates followed by Western blot analysis using TRPC-specific, affinity-purified antibodies revealed the presence of TRPC1, -C3, and -C6. TRPC4, -C5, and -C7 were nondetectable. TRPC1 immunofluorescence was detected in glomeruli and specific tubular cells of the cortex and outer medulla. TRPC1 colocalized with aquaporin-1, a marker for proximal tubule and thin descending limb, but not with aquaporin-2, a marker for connecting tubule and collecting duct cells. TRPC3 and -C6 immunolabeling was predominantly confined to glomeruli and specific tubular cells of the cortex and both the outer and inner medulla. TRPC3 and -C6 colocalized with aquaporin-2, but not with the Na /Ca 2  exchanger or peanut lectin. Thus TRPC3 and -C6 proteins are expressed in principle cells of the collecting duct. In polarized cultures of M1 and IMCD-3 collecting duct cells, TRPC3 was localized exclusively to the apical domain, whereas TRPC6 was found in both the basolateral and apical membranes. TRPC3 and TRPC6 were also detected in primary podocyte cultures, whereas TRPC1 was exclusively expressed in mesangial cell cultures. Specific immunopositive labeling for TRPC4, -C5, or -C7 was not observed in kidney sections or cell lines. These results suggest that TRPC1, -C3, and -C6 may play a functional role in PLC-dependent signaling in specific regions of the nephron. - P. j/ \0 y4 T; L
      【关键词】 ion channels renal nephron immunoprecipitation immunofluorescence subcellular localization polyclonal antibodies4 u& ]- V9 y4 T% |  s$ N
   THE ACTIVITY OF THE TRANSIENT receptor potential (TRP) channel in Drosophila is rapidly increased during photoreception following light-induced activation of PLC (for recent comprehensive review see Ref. 20 ). Drosophila TRP is the founding member of what is now known to be one of the largest channel superfamilies ( 40 ). Of the 34 family members, the TRPC channel subfamily exhibits the greatest homology to the Drosophila homologs and appears to be activated in response to stimulation of G protein-coupled membrane receptors (GPCR) linked to PLC. There are seven members of the mammalian TRPC family, designated TRPC1-TRPC7, but in humans TRPC2 is a pseudogene. The TRPC proteins form Ca 2  -permeable, nonselective cation channels, and as such, are thought to play an important role in Ca 2  signal transduction ( 22 ). TRPC channels may function as homotetramers or heteromultimers ( 9, 11, 31 ), but the actual subunit composition of the native TRPC channels remains essentially unknown. In the Drosophila eye, TRP channels exist in a signaling complex held together by a scaffolding protein called INAD ( 3, 14, 28, 34 ). A similar TRPC "signalplex" may exist in mammalian cells, but again, the number or identity of channel regulatory subunits has not been defined. However, there is growing evidence that the mechanism of channel activation following stimulation of PLC may depend on the subunit composition, the presence of specific accessory proteins, and the targeting of the channels within specific subcellular microdomains ( 4, 24 ). Thus the first step in understanding the impact of TRPC channel function at the organ level is to determine cell and subcellular localization, evaluate native subunit composition, and identify native accessory proteins. Traditionally, the approach would be to address these issues for each channel individually. However, most tissues express multiple forms of the TRPC channel proteins ( 8, 26 ) and numerous splice variants have been described. Furthermore, the potential for heteromultimerization requires the simultaneous evaluation of all family members.$ O: `4 [/ }4 F0 B5 p

Essentially, all cells of the kidney have the capacity to respond to hormones, growth factors, and paracrine substances through GPCRs linked to PLC and downstream activation of Ca 2  signaling events. In each cell type found in the nephron, the rise in intracellular Ca 2  concentration ([Ca 2  ] i ) initiated by receptor stimulation is linked to a specific cellular function. For example, stimulation of mesangial cells of the glomerulus by angiotensin II will increase [Ca 2  ] i and cause contraction with associated changes in capillary loop blood flow and glomerular filtration ( 30 ). Increases in [Ca 2  ] i of the podocyte following stimulation by bradykinin or angiotensin II will influence contractile proteins of the podocyte foot structures and modulate filtration via the slit diaphragm ( 23 ). Activation of proximal tubule cells by low concentrations of parathyroid hormone will increase [Ca 2  ] i ( 33 ), which presumably plays a role in Ca 2  and phosphate homeostasis. Vasopressin has been shown to induce Ca 2  influx across the apical membrane of renal collecting duct cells, and the subsequent alterations in [Ca 2  ] i affect Na reabsorption in these tubules ( 7, 29, 35 ). There are receptors for thrombin, endothelin, EGF, TNF, PDGF, atrial natriuretic factor, leukotrienes, and thromboxanes to list a few, which are linked to PLC in various cell types of the nephron. Furthermore, the constant osmotic challenge posed by the preurine to the tonicity of the tubular epithelia necessitates regulation of cell volume. Again, Ca 2  signaling is important in this process ( 32, 38 ). Thus a possible and perhaps critical role for TRPC channels in glomerular filtration, salt and water retention, pH balance, and control of renal vascular resistance seems likely, but a comprehensive examination of TRPC channels in different regions of the nephron has not been performed. Wang et al. ( 36 ) examined the expression of TRPC channels in a mouse mesangial cell line (MMC). Using RT-PCR, these investigators found message for TRPC1 and TRPC4, which was confirmed at the protein level by Western blot and immunofluorescence analysis. Similarly, kidney sections stained positive for TRPC4 immunoreactivity in the glomerulus. Facemire et al. ( 5 ) found a low level of TRPC1, -C3, -C4, -C5, and -C6 immunoreactivity in glomeruli, whereas preglomerular resistance vessels expressed high levels of TRPC1, -C3, and -C6 relative to that found in the aorta. In a study by Lee-Kwon et al. ( 15 ), TRPC4, but not TRPC5, mRNA and immunoreactivity were detected in rat renal medullary descending vasa recta. Bandyopadhyay et al. ( 1 ) found TRPC1, TRPC3, and TRPC6 in polarized Madin-Darby canine kidney cells in culture. TRPC3 was localized to the apical membrane, TRPC1 was found primarily in the basolateral membrane, and TRPC6 was present in both locations. Finally, two recent studies ( 25, 37 ) have implicated TRPC6 in glomerulosclerosis and reported protein expression in glomerulus and isolated podocytes in culture. To date, these are the only published reports of TRPC channel expression in cells of the kidney.

The purpose of the present study was to determine the distribution of TRPC channels in different regions of rat kidney. Using antibodies directed against two different epitopes in each TRPC channel protein, we were unable to detect the presence of TRPC4, -C5, or -C7. However, immunofluorescence analysis and immunoprecipitation followed by Western blot analysis showed that TRPC1, TRPC3, and TRPC6 channels are differentially expressed in various nephron segments, suggesting that these channels play an important role in Ca 2  signaling events in these regions.- V6 D0 ]' d4 R2 q$ U' d4 R

METHODS$ w3 A  y$ O  j; \

Antibodies. Affinity-purified rabbit polyclonal antibodies specific for each TRPC channel protein were generated and characterized as previously described ( 9 ) (see Supplementary MATERIALS AND METHODS; all supplementary material in this paper can be found at http://ajprenal.physiology.org/cgi/content/full/00376.2005/DC1 ). Commercial antibodies used were from the following sources: mouse anti-aquaporin-1 (AQP1) and goat anti-aquaporin-2 (AQP2; Santa Cruz Biotechnology); Alexa 488-, 594-, and 697-conjugated anti-rabbit, anti-mouse, and anti-goat IgG (secondary antibodies Molecular Probes); mouse anti-Na -K -ATPase (NKA; 1 -subunit; Upstate Biotechnology); mouse anti-Na /Ca 2  exchanger (NCX; Swant); and mouse anti-peanut lectin (Sigma)./ I; W: Y5 X& {" P2 T0 V9 `; K( o
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Cell culture. M1 and IMCD3 cells were obtained from the American Type Culture Collection and cultured in a 1:1 mixture of DMEM and Ham?s F-12 medium containing 2.5 mM L -glutamine, 1% penicillin-streptomycin-neomycin solution (GIBCO), 15 mM HEPES, 0.5 mM Na-pyruvate, and 1.2 g/l Na-bicarbonate at 37°C. M1 cells were supplemented with 0.005 mM dexamethasone, 5% heat-inactivated FBS, whereas IMCD3 cells were supplemented with 10% FBS. Rat glomerular podocytes and mesangial cells, isolated and cultured as previously described ( 10, 27 ), were kindly provided by Dr. John R. Sedor (Case Western Reserve Univ.). Spodoptera frugiperda (Sf9) cells were obtained from American Type Culture Collection and cultured as previously described ( 12, 13 ) using Grace?s Insect Medium supplemented with 2% lactalbumin hydrolysate, 2% yeastolate solution, 2 mM L -glutamine, 10% FBS, and 1% penicillin-streptomycin-neomycin.

Cells on permeable supports. M1 and IMCD3 cells were plated at a density of 1 x 10 5 /well and grown to confluence on 24-mm Transwell culture chambers (Corning Costar). Resistance measurements were made with a Millicell ERS epithelial volt-ohmmeter (Millipore, Allen, TX) as described by the manufacturer.  l( j3 \4 z3 ?

Preparation of rat kidney lysates. Kidneys isolated from adult Sprague-Dawley rats were minced and suspended in lysis buffer containing 150 mM NaCl, 10 mM Tris·HCl (pH 7.5), and 0.1% deoxycholate. The tissue suspension was homogenized on ice using a Brinkman PT10/35 Polytron fitted with a 10-mm generator (3 x 10-s pulses at a power setting of 5). Homogenates/lysates were centrifuged at 6,000 g for 10 min at 4°C to remove tissue debris. The resulting supernatant was incubated at 4°C for 30 min and subsequently subjected to centrifugation at 100,000 g for 60 min at 4°C to remove unsolubilized membrane fragments. The resulting supernatants/lysates were used immediately for immunoprecipitation experiments. All experimental protocols involving the use of animals were approved by and performed in compliance with the Case Western Reserve University Institutional Animal Care and Use Committee guidelines.
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Isolation of glomeruli and tubules. Kidneys were removed from adult Sprague-Dawley rats, and the cortex and medulla were isolated by dissection. Cortex, minced into 1-mm 3 pieces, was gently pressed through a 106-µm cell strainer using a flattened pestle. The filtrate was passed through a new 100-µm cell strainer. The filtrate was collected and passed through a 70-µm cell strainer. The tubule fraction was passed through the strainer and suspended in 5 ml of buffer containing 137 mM NaCl, 2.7 mM KCl, 1 mM Na 2 HPO 4, and 10 mM HEPES (HBSS) and centrifuged at 12,000 g for 30 min at 4°C. The resulting tubule pellet was resuspended in lysis buffer. Glomeruli, which accumulated on the 70-µm strainer, were washed with 5 ml of HBSS and collected. The glomerular suspension, which by microscopic inspection was 90% glomeruli, was subjected to centrifugation at 200 g for 5 min. The supernatant was discarded, and the glomerular pellet was resuspended in lysis buffer.
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Immunoprecipitations and immunoblots. Tissue lysates were precleared by adding control IgG together with protein A/G agarose beads for 1 h at 4°C. Precleared lysates were incubated with different TRPC-specific antibodies, and the immunocomplexes were captured by incubation with protein A/G agarose beads at 4°C for 12 h. Beads were pelleted, washed four times with lysis buffer, resuspended in 100 µl of 2 x SDS sample buffer, and boiled for 3 min. Cell lysates and immunoprecipitated proteins were fractionated by SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes (100 V for 1 h) in Tris-glycine buffer. Blots were probed with the indicated primary antibody and detected, following incubation with horseradish peroxidase-conjugated anti-rabbit IgG, by a SuperSignal West Pico chemiluminescent substrate (Pierce). The figures show representative results from experiments repeated at least three times.

Immunofluorescence. Frozen sections (6 µm) from adult rat kidneys were fixed in paraformaldehyde for 30 min. The sections were briefly rinsed in PBS and subsequently incubated with blocking solution containing 3% IgG-free BSA (Vector Laboratories), 10% normal donkey serum, and 0.1% Triton X-100 for 1 h at room temperature. Sections were incubated with the indicated primary antibody overnight at 4°C. After being washed three times for 5 min with PBS at room temperature, the sections were incubated with Alexa 488- or Alexa 594-conjugated anti-rabbit IgG for 1 h at room temperature. Sections were washed three times with PBS for 5 min and mounted with Prolong Gold antifade medium (Molecular Probes). Images were acquired using a Leica DMIRE2 fluorescence microscope equipped with a computer-controlled mechanical stage and a SPOT RT camera. To obtain high-resolution dual-fluorescence images of the entire kidney cross section, filter cube selection, stage movement, and image acquisition were controlled by Simple PCI software (Compix). For each fluorophore, a montage of 110 images (using a x 10 objective) was created for reconstruction of each cross section. Where indicated, confocal images were acquired using a Leica TCS SP2 confocal microscope with either a x 40 or x 100 objective. Total magnification is given in each figure.

RESULTS AND DISCUSSION

Distribution of TRPC channels in rat kidney. In a previous report, anti-peptide antibodies specific for each TRPC channel protein were generated and characterized ( 9 ). These original antibody preparations have been designated A -TRPCx. In the present study, a second set of antibodies, designated B -TRPCx, was generated in rabbits against a different peptide sequence in each rat TRPC channel protein (Supplementary Table 1). The complete characterization of the TRPC antibodies with regard to specificity, selectivity, and usefulness as probes for immunoprecipitation, immunoblot, and immunohistochemical analysis can be found in the Supplementary Materials.
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The overall approach used to determine the presence and localization of each TRPC channel protein in the kidney was as follows. The presence of each channel was first determined by immunoprecipitation followed by Western blot analysis. This was followed by immunofluorescence analysis of whole kidney cross sections, which provided the initial indication of localization within the nephron. Next, colocalization of each identified TRPC channel protein with specific marker proteins was used to localize the TRPCs to specific cell types within the nephron. Finally, comparison of TRPC subcellular distribution with that of the NKA pump was used to determine localization to apical or basolateral membranes. Once the in vivo distribution was established, we selected several cell lines from different regions of the nephron to examine TRPC channel expression for future use as in vitro model systems. With noted exceptions (differences between A - and B -antibodies are noted in all sections, except the last section where no differences were found), all of the biochemical and immunohistochemical results presented in this paper have been confirmed using both the A -TRPCx and B -TRPCx antibodies. With regard to controls, incubation of kidney tissue and cell lysates with protein A/G beads alone (i.e., no primary antibody) or preincubation of the primary antibodies with the immunizing peptide prevented pull-down of the channel proteins as determined by subsequent immunoblots. For immunofluorescence assays, preincubation of the primary antibodies with the appropriate immunizing peptide, or incubation of kidney sections with only the secondary antibody, reduced fluorescence to nondetectable levels. These controls confirm that the results presented below are specific.9 B% s8 @: n! ^3 S; e0 g

With the use of both A -TRPCx and B -TRPCx antibody preparations, TRPC4, TRPC5, and TRPC7 were nondetectable in immunoprecipitation or immunohistochemical assays. This result was obtained in vivo in rat, dog, and mouse kidney, in cultured rat podocyte and mesangial cells, and in cultured mouse M1 and IMCD3 collecting duct cells. Our inability to detect these channels may indicate that 1 ) their expression level is below the limit of our detection, 2 ) the epitopes recognized by these antibodies are hidden/blocked in native tissues, or 3 ) the epitopes are missing due to splice variant expression. It seems unlikely, however, that the epitopes for both A - and B -antibodies would be blocked or missing in these proteins. Furthermore, the A -antibodies have been used for identification of TRPC channels in rat brain ( 2, 9 ). Thus TRPC4, TRPC5, and TRPC7 channels are probably absent or below the limit of detection in the kidney. Three reports of TRPC4 protein in renal tissue have been published to date ( 5, 15, 36 ). These studies employed commercially available antibody preparations for the identification of TRPC4 protein. However, it has recently been shown that the anti-TRPC4 antibody from Alomone Labs (Jerusalem, Israel) recognizes a protein with the appropriate molecular mass in lysates from TRPC4 knockout mice ( 6 ). Thus results with this antibody should be viewed with caution. TRPC4, -C5, and -C7 will not be discussed further.5 z# n/ E/ z) [

Distribution of TRPC1 in the kidney. Kidney lysates were subjected to immunoprecipitation using both A -TRPC1 and B -TRPC1. As seen in Fig. 1 B, a single band at a molecular mass expected for full-length TRPC1 was immunoprecipitated and recognized by both TRPC1 antibodies. To determine which cells express TRPC1, cross sections of rat kidney were stained with both A - and B -TRPC1 antibodies. In contrast to the immunoprecipitation and Western blot results, only B -TRPC1 yielded labeling profiles consistent with specific cell-associated immunoreactivity in the kidney. Immunofluorescence analysis using B -TRPC1 showed, however, that TRPC1 was present in glomeruli, and specific tubules of the renal cortex and outer medulla, but was essentially absent from the inner medulla ( Fig. 1 A ). To begin to determine the exact location of TRPC1 in the nephron, we compared the distribution of TRPC1 with that of AQP1, which is expressed in regions of the proximal tubule and descending thin limb, and with AQP2 which is found in connecting tubule (CNT) and collecting duct (CD) cells ( 21 ). No colocalization was observed with AQP2 (Supplementary Fig. 4 ); however, colabeling of TRPC1 (green) with AQP1 (red) indicated nearly perfect colocalization (yellow) in tubular elements throughout the cortex ( Fig. 2 ). The high-magnification merged image shows that TRPC1 is highly expressed in the apical brush-border regions of the proximal tubule cells along with AQP1. The AQP1 labeling in the inner medulla presumably reflects the thin descending limb. Thus TRPC1 expression appears to be restricted to the glomerulus and proximal tubule. However, as seen in Fig. 2, the outer stripe of the inner medulla is predominantly green; i.e., TRPC1 is expressed in tubules that do not express either AQP1 or AQP2.9 P% _6 N" \# ~8 A

Fig. 1. Localization of transient receptor potential TRPC1 channels in cortex and medulla of rat kidney. A : total lysates, prepared from rat kidney as described in METHODS, were subjected to immunoprecipitation followed by Western blot analysis. The antibodies used for immunoprecipitation (IP) and immunoblotting (Blot) are indicated below each lane. B : fluorescence images of rat kidney sections were labeled with the B -TRPC1 antibody. In this and all subsequent figures, kidney cross-sectional images ( left ) were obtained by conventional fluorescence microscopy as described in METHODS. Higher-magnification images ( right ) were obtained by confocal microscopy.

Fig. 4. Localization of TRPC6 channels in cortex and medulla of rat kidney. A : lysates were prepared from medulla and from glomerular and tubular fractions from rat kidney as described in METHODS. Lysates were subjected to immunoprecipitation followed by Western blot analysis. The antibodies used for immunoprecipitation (IP) and immunoblotting (Blot) are indicated below each lane. B : fluorescence images of rat kidney sections were labeled with the A -TRPC6 antibody.' M# I9 Y, B- v" H- M& o
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Fig. 2. Colocalization of TRPC1 channels with aquaporin-1 (AQP1) in rat kidney. Fluorescence images of rat kidney sections were colabeled with B -TRPC1 (green) and anti-aquaporin-1(AQP1) antibodies (red).; V& K2 a5 k) t5 ~! H& J
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Distribution of TRPC3 and TRPC6 in the kidney. For these experiments, the medulla was separated from the cortex by dissection and the cortex was separated into glomerular and tubular fractions using a sieving protocol as described in METHODS. As seen Fig. 3, a single immunoreactive band of 95 kDa was observed in immunoprecipitates from medullary lysates. Both antibodies immunoprecipitate and recognize the same protein in the medulla. Immunoprecipitations from glomerular and tubular lysates from the cortex also showed the same 95-kDa band with both the A - and B -TRPC3. However, an additional higher-molecular-weight band at 120 kDa was observed when the immunoprecipitated proteins were probed with B -TRPC3 ( Fig. 3 A, bottom right ), suggesting the possible presence of an additional splice variant of TRPC3 channel protein. According to a recent report ( 39 ), there is a TRPC3 splice variant (TRPC3a) with an extra exon at the 5'-end of the gene, adding an additional 62 amino acids to the murine homolog. However, the observation that this band is not recognized by the A -TRPC3 antibody on Western blot analysis suggests that it may be unrelated to TRPC3. Distinct immunopositive labeling for TRPC3 was detected in both the cortical and medullary regions of the kidney ( Fig. 3 ). In both the cortex and medulla, TRPC3 localization was predominantly confined to the apical domain of specific tubular cells. In the cortex, TRPC3 was localized to the glomeruli. This is consistent with a recent study reporting TRPC3 mRNA in isolated glomeruli ( 5 ). Given the results obtained by Western blot analysis, it is important to reemphasize that the same immunohistochemical profile was observed using both the A -TRPC3 and B -TRPC3 antibodies.! ~, @& I$ ]6 Z) E
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Fig. 3. Localization of TRPC3 channels in cortex and medulla of rat kidney. A : lysates were prepared from medulla and from glomerular and tubular fractions from rat kidney as described in METHODS. Lysates were subjected to immunoprecipitation followed by Western blot analysis. The antibodies used for immunoprecipitation (IP) and immunoblotting (Blot) are indicated below each lane. B : fluorescence images of rat kidney sections were labeled with the A -TRPC3 antibody.+ u, H" v) x5 R3 h, p# H

Immunoprecipitations from the medulla using both A -TRPC6 and B -TRPC6 showed an immunoreactive band of 110 kDa ( Fig. 4 ). However, immunoprecipitations from glomeruli showed the presence of an immunoreactive TRPC6 band of 90 kDa. According to a previous report ( 41 ), rats express full-length and two splice variant forms of TRPC6, with predicted molecular masses of 107, 98, and 87 kDa. Thus it is possible that cells of the glomerulus predominantly express one of the shorter TRPC6 splice variants, whereas the full-length form predominates in tubular cells of the renal cortex and medulla. Immunofluorescence analysis revealed that TRPC6 channel protein distribution was similar to that of TRPC3. TRPC6 was present in the glomerulus and in specific cells of the cortex and both the outer and inner medulla ( Fig. 4 ). Association of TRPC6 with cells of the glomerulus is consistent with recent reports demonstrating a role for TRPC6 in focal segmental glomerulosclerosis ( 25, 37 ). In high-magnification images of tubule cells, TRPC6 appeared to localize to both the apical and basolateral domains.( j7 A3 o4 T" X4 C& k8 T! {

Kidney sections were individually colabeled for TRPC3 or TRPC6 and AQP1 or AQP2. Neither TRPC3 nor TRPC6 localized with AQP1 (Supplementary Fig. 5 ). However, both proteins were found in cells expressing AQP2 ( Fig. 5 ). AQP2 is abundant in the apical region of the principal cells of the CD and at lower abundance in cells of the CNT ( 21 ). At higher magnification, TRPC3 labeling appeared predominantly at the apical membrane, although some intracellular labeling is evident in most images. TRPC6 immunofluorescence was detected in both the apical and basolateral membranes and, like TRPC3, may also be present in the cytosol. To distinguish between CNT and CD, we compared the distribution of TRPC3 and TRPC6 with the NCX. The NCX is present in distal convoluted tubule and in CNT, but its expression abruptly stops in the cortical CD ( 17, 18 ). Consistent with this distribution, NCX immunofluorescence was seen in the cortex, but was absent from the inner medulla ( Fig. 6 ). Neither TRPC3 nor TRPC6 colocalized with NCX. Thus TRPC3 and TRPC6 appear to be highly expressed in cells of the cortical and medullary CD. The CD is made up of two primary cell types, principle cells and intercalated cells. The principle cells express AQP2 and are thought to be involved in vasopressin-regulated water transport, whereas the intercalated cells express the H -ATPase and are thought to play a role in acid secretion ( 19 ). Peanut lectin is a marker for intercalated cells ( 16 ). Neither TRPC3 nor TRPC6 colocalized with peanut lectin (data not shown), suggesting that both channel proteins are primarily expressed in the principle cells of the CD.
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Fig. 5. Colocalization of TRPC3 and TRPC6 channels with aquaporin-2 (AQP2) in rat kidney. Fluorescence images of rat kidney sections were colabeled with A -TRPC3 or A -TRPC6 (green) and anti-AQP2 antibodies (red) as indicated in each panel.

Fig. 6. Colocalization of TRPC3 and TRPC6 channels with the Na /Ca 2  exchanger (NCX) in rat kidney. Fluorescence images of rat kidney sections were colabeled with A -TRPC3 or A -TRPC6 (green) and anti-NCX antibodies (red).

To further examine the subcellular localization of TRPC3 and TRPC6, kidney cross sections were individually costained for either TRPC3 or TRPC6 and NKA, which is confined to the basolateral membrane of most renal epithelial cells. As seen in Fig. 7, TRPC3 (green) is present in the apical region, whereas NKA (red) is present in the basolateral domain in cells of the CD. No colocalization (yellow) of TRPC3 and NKA was observed in merged images. In contrast, TRPC6 (green) and NKA (red) immunofluorescence showed substantial overlap (yellow) in high-magnification images ( Fig. 7 ). This result is consistent with our recent study showing that native NKA coimmunoprecipitates with native TRPC6 from kidney and from HEK cells heterologously expressing TRPC6. TRPC1 did not colocalize with the NKA and appears to be exclusively expressed in the apical membrane of proximal tubule cells ( Fig. 7 ).6 H& P: [+ V% C6 o3 Z

Fig. 7. Colocalization of TRPC1, TRPC3, and TRPC6 channels with Na -K -ATPase in rat kidney. Fluorescence images of rat kidney sections were colabeled with B -TRPC1, A -TRPC3, or A -TRPC6 (green) and anti-Na -K -ATPase antibodies (red).

Expression of TRPC1, -C3, and -C6 in kidney cell lines. Podocytes and mesangial cells from the glomerulus, and M1 cortical CD cells and IMCD3 intermedullary CD cells, were examined for the presence of TRPC channel proteins. The various cell lines were grown as nonpolarized cultures on plastic tissue culture dishes (for immunoprecipitation) or on glass coverslips (for immunofluorescence analysis). As seen in Fig. 8 A, mesangial cells expressed TRPC1, whereas podocytes, M1, and IMCD3 cells expressed TRPC3 and TRPC6. In contrast to the in vivo results (see Fig. 3 ), only a single protein band at a molecular mass expected for full-length TRPC3 was observed in podocytes, M1, and IMCD3 cells. However, as seen in Fig. 8, B and D, the shorter form of TRPC6 was found in podocytes, whereas the longer form was exclusively expressed in M1 and IMCD3 cells. These results are consistent with the in vivo observations (see Fig. 4 ) and provide additional support for the hypothesis that mutations in a splice variant form of TRPC6 may be responsible for glomerulosclerosis.1 n3 ^3 e" P& \8 H7 a: ]+ r/ u
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Fig. 8. Immunoprecipitation of TRPC1, TRPC3, and TRPC6 channel proteins from kidney-derived cell lines. Lysates were prepared from mesangial cells ( A ), podocytes ( B ), or M1 and IMCD3 cells ( C and D ). The antibodies used for immunoprecipitation (IP) and immunoblotting (Blot) are indicated below each lane.$ T* V6 l" `, g" J
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Immunofluorescence analysis showed the presence of TRPC3 in both M1 and IMCD-3 cells ( Fig. 9 ). In nonpolarized M1 cells, TRPC3 labeling appeared predominantly in the plasma membrane, whereas in IMCD-3 cells labeling was more intracellular. A similar profile of labeling was seen for TRPC6 in both nonpolarized M1 and IMCD3 cells ( Fig. 10 ). To determine localization in polarized cells, both M1 and IMCD-3 cells were grown on Transwell filters, and TRPC3 and TRPC6 immunofluorescence was evaluated using confocal microscopy. As seen in Fig. 9, TRPC3 (green) was apically localized in both M1 and IMCD3 cells and no colocalization was observed with NKA in the basolateral membrane as shown in the x - z plane image. TRPC6 was also observed in the apical membrane of both cell types but also showed substantial colocalization with NKA in the basolateral membrane. These results are consistent with a recent study that found TRPC3 exclusively localized to the apical membrane of polarized Madin-Darby canine kidney cells, whereas TRPC6 was present in both the apical and basolateral domains ( 1 ).
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Fig. 9. Localization of TRPC3 channels in polarized M1 and IMCD-3 cells. A -TRPC3 antibody was used to detect the presence of endogenous protein in M1 and IMCD-3 cells by immunofluorescence. A and B : confocal images from nonpolarized cells. C - F : confocal images from polarized M1 and IMCD3 cell monolayers colabeled with A -TRPC3 and anti-Na -K -ATPase antibodies. C and D : x - y images of TRPC3 (green). E and F : x - y images of Na -K -ATPase (red) in the 2 cell types. Middle : high-resolution line scan in the x - z plane with the apical membrane surface oriented up.
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Fig. 10. Localization of TRPC6 channels in polarized M1 and IMCD-3 cells. A -TRPC6 antibody was used to detect the presence of endogenous protein from M1 and IMCD-3 cells by immunofluorescence. A and B : confocal images from nonpolarized cells. C - F : confocal images from polarized M1 and IMCD3 cell monolayers colabeled with A -TRPC6 and anti-Na -K -ATPase antibodies. C and D : x - y images of TRPC6 (green). E and F : x - y images of Na -K -ATPase (red) in the 2 cell types. Middle : high-resolution line scan in the x - z plane with the apical membrane surface oriented up.
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Immunofluorescence analysis showed that mesangial cells express TRPC1 ( Fig. 11 ). The labeling was primarily restricted to the plasma membrane. In contrast, podocytes expressed both TRPC3 and TRPC6. Labeling was again seen in the plasma membrane, but was also intracellular. The intracellular labeling appeared in tracks. At high magnification, deconvoluted confocal images revealed punctate TRPC3 immunofluorescence aligned in tracks radiating out from the perinuclear region of the podocyte, reminiscent of cargo transport via the tubulovesicular pathway. A similar, although less distinct, punctate profile was observed for TRPC6 immunofluorescence in the podocyte.

Fig. 11. Localization of TRPC1, TRPC3, and TRPC6 channels in kidney-derived cell lines. B -TRPC1 antibody was used for immunofluorescence ( top ) in cultured rat mesangial cells. A -TRPC3 ( middle ) and A -TRPC6 ( bottom ) were used for immunolabeling of podocytes isolated from rat kidney. Areas indicated by white rectangles are shown enlarged on the right.$ z* ]7 w9 H1 x* _
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In conclusion, TRPC1 channel protein is expressed in the glomerulus and in the apical membrane of proximal tubule cells where it colocalizes with AQP1. Additionally, TRPC1 was found in cultured mesangial cells. TRPC3 and TRPC6 channel proteins exhibited nearly identical cell-specific distribution in the nephron. TRPC3 and TRPC6 were localized to the glomerulus and to principal cells of the CD, where they colocalize with AQP2. Consistent with the in vivo distribution, only TRPC3 and TRPC6 were found in cultured M1 and IMCD3 cell lines. TRPC3 was localized to the apical membrane both in vivo and in cultured cells, whereas TRPC6 was found in both the apical and basolateral membranes. The selective and differential localization of TRPC1, TRPC3, and TRPC6 suggest that these channels play a specific role in PLC-dependent responses within the kidney. Our next goal will be to determine the actual subunit composition and to begin isolation of specific binding partners from these regions.

GRANTS' s! r: Y+ V6 G
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This study was supported by National Institute of General Medical Sciences Grant GM-52019.

ACKNOWLEDGMENTS  S+ v" R: ]( I: N6 T( w
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We thank Dr. Martha Konieczkowski for helpful discussion concerning isolation of glomeruli and Pat Glazebrook for help with the confocal imaging.$ L5 D4 m1 J& Z3 {
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