Expression of TRPC4 channel protein that interacts with NHERF-2 in rat descending vasa recta

Whaseon Lee-Kwon,1 James B. Wade,2 Zhong Zhang,1 Thomas L. Pallone,1,2 and Edward J. Weinman1,2,3

Departments of 1Medicine and 2Physiology, University of Maryland School of Medicine; and 3Department of Veterans Affairs Medical Center, Baltimore, Maryland

Submitted 24 August 2004 ; accepted in final form 6 December 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The PDZ domain adaptor protein Na+/H+ exchanger regulatory factor (NHERF)-2 is expressed in renal medullary descending vasa recta (DVR), although its function has not been defined. Transient receptor potential channels (TRPC) TRPC4 and TRPC5, nonselective cation channels that transport Ca2+, were recently demonstrated to complex with the NHERF proteins. We investigated whether TRPC4 and/or TRPC5 are associated with NHERF-2 in DVR. RT-PCR revealed mRNA for TRPC4 and NHERF-2, but not for TRPC5 or NHERF-1, in microdissected DVR. Immunohistochemical studies demonstrated expression of TRPC4 and NHERF-2 proteins in both the endothelial cells and pericytes. These proteins colocalized in some cells of the DVR. TRPC4 coimmunoprecipitated with NHERF-2 from renal medullary lysates, and NHERF-2 coimmunoprecipitated with TRPC4. TRPC5 was not detected in DVR with the use of immunohistochemistry or in NHERF-2 immunoprecipitates. We conclude that DVR pericytes and endothelia coexpress TRPC4 and NHERF-2 mRNA and protein and that these proteins colocalize and coimmunoprecipitate, indicating a possible physical association. These findings suggest that TRPC4 and NHERF-2 may play a role in interactions related to Ca2+ signaling.

PDZ proteins; calcium channels; medulla; pericytes; endothelium; microcirculation


DESCENDING VASA RECTA (DVR) are capillary-sized (10–18 µm) resistance vessels that perfuse the renal medulla. They originate as branches of juxtamedullary efferent arterioles and traverse the outer medulla in vascular bundles from which they supply blood to both the outer and inner medullae. Their radial distribution within vascular bundles and their contractility imply a probable role in the regulation of regional perfusion within the medulla (6, 15). DVR are lined with a continuous endothelium that generates nitric oxide (NO) and are enveloped by smooth muscle-like pericytes that impart contractility (1215, 17, 18). Both NO production and contraction of smooth muscle cells are regulated by changes in the cytoplasmic concentration of Ca2+. On the basis of these considerations, we infer that Ca2+ signaling processes within DVR pericytes and endothelia are important regulators of medullary perfusion, which, in turn, has been associated with tissue oxygenation, salt excretion, abrogation of acute renal failure, and the urinary concentrating mechanism (24, 7, 1215).

In prior experiments, we investigated Ca2+ signaling pathways and the channel architecture of DVR pericytes and endothelia. Angiotensin II depolarizes pericytes by activating a Ca2+-dependent Cl conductance, leading to Ca2+ entry into the cytoplasm and DVR contraction (13, 15, 29). Interestingly, angiotensin II inhibits Ca2+ signaling in DVR endothelial cells (18). Vasodilators such as acetylcholine and bradykinin as well as wall stress increase the cytosolic concentration of Ca2+ and NO production in endothelial cells (15, 27). The pathways that mediate Ca2+ entry into the cytoplasm of these cells is, at present, poorly defined.

The two isoforms of the Na+/H+ exchanger regulatory factor (NHERF-1 and NHERF-2) are scaffolding proteins that facilitate protein-protein interactions through their tandem PDZ-binding domains and their COOH-terminal ezrin-radixin-moesin-merlin binding domain (19, 22, 26). We previously identified the presence of NHERF-2 in the DVR of the rat kidney, although its explicit function is unknown (24). Recent studies have suggested that some members of the transient receptor potential (TRP) protein family function as nonselective cation channels that conduct Ca2+ and Na+ ions into the cytoplasm (5, 9, 10). Two members of the canonical subclass of the TRP family of proteins, TRP channels (TRPC) TRPC4 and TRPC5, have a COOH-terminal amino acid sequence, TRL, that represents a class 1 PDZ-binding domain. With the use of a heterologous expression model, recent studies have indicated that NHERF binds TRPC4 and TRPC5 and directs its expression in the plasma membrane (20). Given NHERF-2 expression in the DVR and the important physiological role of Ca2+ in the function of DVR, we initiated experiments to explore the possibility that TRPC4 and/or TRPC5 are expressed in DVR pericytes and/or endothelial cells and to determine whether these Ca2+ channels interact with NHERF. The results show that both the pericytes and endothelia of DVR express TRPC4 and NHERF-2 but not TRPC5. Confocal microscopy and coimmunoprecipitation experiments verified that TRPC4 and NHERF-2 are physically associated, findings that suggest a functional interaction.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
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Isolation of DVR. All investigations were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland. Female Sprague-Dawley rats (70–150 g; Harlan) were anesthetized by administration of an intraperitoneal injection of thiopental (50 mg/kg body wt). Kidneys were perfused with solution A containing (in mM) 135 NaCl, 1 KCl, 0.1 Na2HPO4, 0.12 Na2SO4, 1.2 MgSO4, 0.3 NaOAc, 5 HEPES, 2.5 CaCl2, and 5.5 glucose, with pH adjusted to 7.4 using NaOH. Microdissection solution for reverse transcriptase (RT)-PCR was prepared by adding 1 mg/ml bovine serum albumin and 1 mg/ml collagenase (type 1), 1 mM DTT, and 1 U/µl RNase inhibitor (Clontech) to solution A. Kidney slices were transferred to a petri dish and maintained at 4°C. Individual DVR, vascular bundles, and selected nephron segments were isolated by microdissection and transferred to a separate petri dish that contained the same microdissection solution as a "wash buffer." The transfer into and then out of the wash buffer served to clear any debris that accompanied the isolated nephron segment or microvessel of interest. Microdissected structures were subsequently transferred from the wash buffer to an RT-PCR reaction tube. Each sample was centrifuged at 10,000 g for 10 min and then rinsed three times with 20 µl of ice-cold microdissection solution. Separate samples of the wash buffer were also analyzed using RT-PCR to rule out contamination during transfer. These controls were uniformly negative.

RT-PCR. RT-PCR was performed using a PCR and cDNA synthesis kit (Invitrogen). Reaction tubes were centrifuged for a few seconds at 10,000 rpm, and the supernatant was discarded. Samples were incubated for 15 min on ice with 2% Triton X-100 containing RNase inhibitor and 5 mM DTT to permeabilize the cells. First-strand synthesis was accomplished by incubating the samples with a RT mixture containing random primers at 42°C for 60 min. The reaction was stopped by heating the sample to 70°C for 15 min. The samples were stored at 4°C until PCR.

PCR for TRPC4 and NHERF-2 was performed using degenerative primers pairs. For TRPC4, the sense primer was 5'-GCCCCCTACCGAGACCGCATCCC-3' (bp 173–206) and the antisense primer was 5'-CCCCACGAGGTCCGCTGTAACTGTG-3' (bp 512–536). For the nested PCR of TRPC4, the sense primer was 5'-CTCTCACCATCAGAGAAAGCCTAC-3' (bp 235–258). TRPC4 antisense primers were 5'-ACTCTTGGTCCAGAAGGGTGTCTC-3' (bp 628–651) and 5'-GGTGCCTCCCATCCTCCTTGACAAA-3' (bp 529–553). For the nested PCR of TRPC5, the sense primer was 5'-CATGGAGCTACTGCTGAACCACAG-3' (bp 255–278) and 5'-GTATGTGGGCGATGCATTACTCTATGCC-3' (bp 281–309); the antisense primers were 5'-GATGACCACAGCGAAGAACTTGACCC-3' (bp 817–842) and 5'-GCTCGGAGCTCC c/a G a/g GAACTGGAG-3' (bp 775–798). The sense primer for NHERF-2 was 5'-CCACAGGATCAAGGCTGTGGAGGGACAG-3' (bp 381–408), and the antisense primer was 5'-GGTCTCCACCTGAGCCCCACAGCAG-3' (bp 1,057–1,081). Nested PCR for NHERF-2 used the sense primer 5'-GCCGGCGGCAACTGACCTGCACTGAGG-3' (bp 452–478) and the antisense primer 5'-GGACAATGAGGATGGCAGTGCTTGG-3' (bp 1,008–1,032). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using 5'-TCCCTCAAGATTGTCAGCAA-3' (bp 1,269–1,289) as the forward primer and 5'-AGATCCACAACGGATACATT-3' (bp 1,558–1,577) as the reverse primer. Each primer (100 µl) was used at 0.2 pmol/µl. The annealing temperature was 56°C for 1 min and the final extension was at 72°C for 7 min.

PCR product analysis. The amplified products were ethanol precipitated and separated by electrophoresis on 1.5% agarose gels. DNA bands were visualized under ultraviolet light after staining with ethidium bromide. The PCR products were subcloned into the pCR II-TOPO vector (Invitrogen) and sequenced (Bio Polymer Lab).

Immunoblotting and immunoprecipitation. Cortical and medullary lysates were prepared from kidneys of the rat. Kidneys were decapsulated, cleaned of connective tissue, and separated into cortex and medulla by dissection. Homogenization and centrifugation were performed to solubilize proteins for Western blot analysis. The protein concentration of the lysates was measured using a bicinchoninic acid (BCA) protein assay kit (Pierce). Aliquots (30 µg) of total lysates were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Proteins were transferred to a nitrocellulose membrane and then exposed to primary antibody. Antibodies for the immunoblots included anti-TRPC4 (1:200 dilution; Chemicon), anti-NHERF-2 (1:500 dilution). Secondary antibodies for immunoblots were conjugated to horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories) at 1:5,000 dilution. Blots were developed with the enhanced chemiluminescence (ECL) kit (Amersham Biosciences) according to the manufacturer's instructions. Coimmunoprecipitation experiments were performed using cortical and medullary lysates. Aliquots (1 mg of protein) of the lysates were reacted with 1 µg of anti-NHERF-2 antibody for 1 h at 4°C. Immune complexes were separated by binding to protein A-Sepharose resin followed by five washes with buffer containing 0.5% Nonidet P-40. Proteins were separated on 10% SDS-PAGE using Tris-glycine-SDS running buffer (Bio-Rad) at 100 V for 2 h, electrophoretically transferred to nitrocellulose membranes, maintained at 30 V overnight at 4°C, and then probed with secondary antibodies as specified above. The reverse experiment also was performed, in which anti-TRPC4 was used for the immunoprecipitation step (1 mg lysate protein) and the immunoprecipitates were probed for TRPC4 (1:200 dilution; Chemicon) and NHERF-2 (1:500 dilution) after separation and transfer.

Immunocytochemical staining of renal medulla and isolated DVR. Tissue sections of the renal medulla of rat kidney were prepared as described previously (23, 24). For direct study of endothelial cells and pericytes, hand-dissected DVR were placed on glass coverslips, fixed with 3% paraformaldehyde in 0.1 M cacodylate for 5 min at 25°C, and washed with microdissection solution. Samples were then incubated with 5% BSA and 0.1% Triton X-100 in microdissection solution for 30 min at 25°C, reacted with primary antibody overnight at 4°C, and washed three times. Reaction with secondary antibodies proceeded for 1 h at room temperature. In some experiments, the primary TRPC4 antibody was the rabbit polyclonal anti-TRPC4 (1:50 dilution; Chemicon). To generate a more specific antibody that functioned at higher titers, a synthetic peptide corresponding to amino acids 946–961 of rat TRPC4 was used to immunize chickens. Serum was affinity purified using the synthetic peptide coupled to keyhole limpet hemocyanine cross linked to agarose beads. Immunostaining of NHERF-2 was performed with a previously characterized rabbit polyclonal antibody (1:100 dilution) (23, 24). Pericytes were identified with monoclonal antibody against {alpha}-smooth muscle actin (1:500 dilution; Sigma). Endothelium was identified using antibody against aquaporin-1 (1:100 dilution; Alomone Laboratories). The secondary antibody was Alexa 488-conjugated donkey anti-rabbit or anti-chicken IgG (1:200 dilution; Molecular Probes). Negative controls were performed by omitting the incubation with primary antibody. Immunofluorescent images were captured with a Zeiss LSM410 confocal fluorescence microscope that uses Coherent INNOVA Enterprise Model 653 and Zeiss He/Ne internal lasers. Images were captured at 512 x 512-pixel resolution and z-axis sectioning at 0.5-µm intervals.

Materials. TRPC4 and TRPC5 antibodies were purchased from Chemicon. Aquaporin-1 antibody was obtained from Alomone Laboratories. Secondary HRP-conjugated antibodies for immunoblotting were purchased from Jackson ImmunoResearch Laboratory. Secondary Alexa-conjugated antibodies for immunofluorescence were obtained from Molecular Probes. Tris-glycine-SDS running buffer was purchased from Bio-Rad, and the BCA protein assay kit was obtained from Pierce Biotechnology. ECL solution and protein A-Sepharose resin were purchased from Amersham. RNase inhibitor was obtained from Clontech. RT-PCR was performed using a kit purchased from Invitrogen.


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
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To determine the distribution of message, nested RT-PCR was performed using hand-dissected DVR and other nephron segments. A 416-bp product representing TRPC4 was readily detected in vascular bundles and isolated DVR as shown in Fig. 1A. TRPC4 was not detected in cells of the thin or thick ascending limbs of the loop of Henle or the collecting ducts. As shown in Fig. 1B, a product representing NHERF-2 also was detected in the DVR and vascular bundles, but not in the collecting duct or the thick ascending limb. NHERF-2 was weakly detected in some but not all proximal tubule segment samples. TRPC5 was not detected in DVR, thick ascending limb, collecting duct, or proximal tubule. GAPDH, run as a control, was present in all samples (data not shown). We next performed Western immunoblot analysis using lysates from the renal cortex and medulla. TRPC4 was detected in both the medullary and cortical lysates (Fig. 2). TRPC5 protein was not detected in the cortex, but a faint band was observed in the medulla. When considered together with the RT-PCR data, these findings suggest that TRPC5 might be expressed in medullary structures other than the DVR.



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Fig. 1. A: nested RT-PCR analysis of transient receptor potential channels (TRPC) TRPC4. Representative results are displayed for hand-dissected descending vasa recta (DVR; lanes 1 and 2), vascular bundles (lanes 3 and 4), thin descending limbs of Henle (lane 5), thick ascending limbs of Henle (lane 6), and collecting ducts (lanes 7 and 8). A 416-bp product representing TPRC4 is identified in DVR and vascular bundles. Lane 9 is the wash buffer control, and lane 10 is without RT. B: nested RT-PCR analysis of PDZ domain adaptor protein Na+/H+ exchanger regulatory factor (NHERF)-2 (574 bp). C: nested PCR of TRPC5 (516 bp). Lanes in B and C are thick ascending limb (lane 1), DVR (lanes 2 and 4), vascular bundle (lane 5), collecting duct (lanes 3 and 6), and proximal tubule (lanes 7–9). Lane 10 is the wash buffer control, and lane 11 is without RT.

 


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Fig. 2. TRPC4 (top) and TRPC5 (bottom) protein expression in renal cortex (lane 1) and medulla (lane 2). Immunoblots were performed using freshly isolated cell lysates prepared from rat cortex and medulla.

 
As shown in Fig. 3, the newly developed chicken antibody directed against amino acids 946–961 of TRPC4 detected a band in medullary lysates, and immunodetection was completely abolished by preincubation with the immunizing peptide. This antibody was subsequently used to study the expression of TRPC4 in the medulla of the kidney.



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Fig. 3. Western immunoblot analysis of renal medullary lysates using an anti-TRPC4 antibody raised in chicken. The antibody recognizes TRPC4 in medullary lysates (lane 1). Preincubation of the antibody with the immunizing peptide abolishes the TRPC4 band (lane 2).

 
Figure 4 shows representative confocal images through the renal medulla that stained for TRPC4 and NHERF-2. Figure 4 shows immunostaining of TRPC4 (Fig. 4A) and NHERF-2 (Fig. 4B) in vascular bundles of the outer medulla. The merged image (Fig. 4C) shows colocalization of the two proteins. Figure 4B shows the presence of TRPC4 and NHERF-2 in outer medullary vascular bundles and DVR (Fig. 4, A and B). TRPC4 and NHERF-2 are also expressed in glomeruli and peritubular capillaries of the cortex (Fig. 4, C and D). Peritubular capillaries outside vascular bundles in the medulla and within the renal cortex also express TRPC4 and NHERF-2 (Fig. 4B).



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Fig. 4. A: representative confocal immunofluorescence microscopic images of the vascular bundle of renal medulla stained for TRPC4 (Aa, red) and NHERF-2 (Ab, green). Ac: merged image of subparts Aa and Ab. Ba and Bb: confocal images of TRPC4 and NHERF-2, respectively, in DVR within vascular bundles of renal medulla, indicated by asterisks. Peritubular capillaries away from the vascular bundles also colabel (pc). Bc and Bd: immunolocalization of TRPC4 and NHERF-2, respectively, in glomeruli and peritubular (pc) capillaries in the renal cortex. Bar, 12 µM.

 
DVR consist of two cell types: pericytes and endothelia. To study the specific expression of TRPC4 and NHERF-2 in those cells, we hand dissected, mounted, and fixed DVR for immunostaining and confocal microscopy. Figure 5 shows expression of {alpha}-smooth muscle actin (Fig. 5A), specific for pericytes, and TRPC4 (Fig. 5B). Figure 5C shows a merged image of Fig. 5, A and B. TRPC4 is expressed in both pericytes and endothelial cells. Pericytes jut out from the abluminal surface, while endothelia line the lumen. Similar images were published previously (12, 13, 15, 28). Figure 6 shows a set of confocal images stained for {alpha}-smooth muscle actin (Fig. 6A) and NHERF-2 (Fig. 6B). Figure 6C shows the merged image of Fig. 6, A and B. Actin and NHERF-2 colocalize in pericytes. NHERF-2 is also expressed in the endothelium. DVR endothelia express the aquaporin-1 water channel (13, 15). To provide an additional visual comparison of markers specific for endothelia and pericytes, Fig. 7 shows immunostaining of DVR using antibodies directed against aquaporin-1 (endothelium) and {alpha}-smooth muscle actin (pericyte). In summary, the confocal images in Figs. 47 indicate colocalization of TRPC4 and NHERF-2 in pericytes and endothelia of DVR from the renal medulla, suggesting a physical association between those proteins.



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Fig. 5. Confocal microscopic images of hand-dissected DVR immunostained for {alpha}-smooth muscle actin and TRPC4. Actin is identified in pericytes (A, red), while TRPC4 is found in both endothelia and pericytes (B, green). C: merged image of A and B. D: white-lighted image of an isolated DVR that was not subjected to fixation.

 


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Fig. 6. Confocal microscopic images of hand-dissected DVR immunostained for {alpha}-smooth muscle actin and NHERF-2. Actin is identified in pericytes (A, red), while NHERF-2 is found in both endothelia and pericytes (B, green). C: merged image of A and B.

 


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Fig. 7. Confocal microscopic images of hand-dissected DVR immunostained for {alpha}-smooth muscle actin and aquaporin-1. Actin is identified in pericytes (A, green), and aquaporin-1 is shown in endothelia (B, red). C: merged image of A and B.

 
To extend these observations and test for a physical association between TRPC4 and NHERF-2, NHERF-2 was immunoprecipitated from lysates of tissue harvested from the renal cortex and medulla, and the immunoprecipitates were probed for the presence of TRPC4 and TRPC5. As shown in Fig. 8A, NHERF-2 immunoprecipitates from the medulla contained significantly more TRPC4 than did immunoprecipitates from the cortex. TRPC5 was not detected by immunoblot in samples from either location (not shown). Figure 8B shows the results of the reverse experiment, in which the rabbit polyclonal antibody directed against TRPC4 was used for immunoprecipitation. Both TRPC4 and NHERF-2 were recovered from the anti-TRPC4 immunoprecipitates, further indicating their physical association.



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Fig. 8. Immunoprecipitation (IP) of NHERF-2 and TRPC4 from lysates of renal cortex (lane 1) and medulla (lane 2). NHERF-2 and TRPC4 in the immune complexes were measured using immunoblot analysis with anti-TRPC4 and anti-NHERF-2. Left: TRPC4 is detected in NHERF-2 immunoprecipitates from cortex and medulla. There was relatively greater recovery of TRPC4 from the medulla compared with the cortex. Right: NHERF-2 is detected in TRPC4 immunoprecipitates.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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It has been established that regulation of renal medullary perfusion is essential for proper function of the urinary concentrating and diluting mechanism and the regulation of salt excretion (24, 6, 7, 12, 15). Blood flow to the medulla is carried by DVR, microvessels that are composed of smooth muscle-like pericytes and capillary endothelia. Recent studies have established that those cells contain functional hormone receptors that activate ion channels and participate in complex signaling pathways that lead to vasoconstriction and release of NO. It is clear that modulation of intracellular Ca2+ plays an important role in the regulation of those processes in DVR pericytes and endothelia (1215, 18, 2729).

The adaptor proteins NHERF-1 and NHERF-2 are expressed in the kidney in selected cells of the nephron, where they are thought to modulate the function of other proteins with which they interact (19, 2224, 26). One remarkable finding, initially observed in the medulla of the rat kidney and recently confirmed in the mouse, was prominent NHERF-2 staining of DVR (1, 23, 24). In the medulla, antibodies directed against NHERF-2 recognized only DVR. NHERF-1 was not detected in DVR or any other structures within the renal medulla. The NHERF proteins contain two PDZ protein-interactive domains and a COOH-terminal sequence that binds to structural proteins in the cell. More than 50 proteins that interact with NHERF isoforms have been identified, including transporters, ion channels, receptors, and signaling proteins (22). Specific binding targets of NHERF-2 in DVR previously were unknown.

The large TRP family of proteins is divided into several subfamilies. The function of many of the individual members is either unknown or poorly characterized. Two members of the canonical subfamily, TRPC4 and TRPC5, have been suggested to function as nonselective cation channels. They have COOH-terminal sequences that posses class 1 PDZ-binding domains (9, 10, 11, 21). The latter is of considerable interest because TRP channels in the Drosophila eye interact with the PDZ domain of INAD (protein responsible for the inactivation no afterpotential Drosophila mutant D) (10, 11). A human homolog of INAD has not been identified, and it has been speculated that NHERF-1 and NHERF-2 might serve as their functional analog in mammals (8, 20). Mery et al. (8) provided evidence that the PDZ-interacting domain of TRPC4 controlled its localization and surface expression in human embryonic kidney HEK-293 cells. Tang et al. (20) demonstrated that TRPC4 and TRPC5, as well as phospholipase C-{beta}1 and phospholipase C-{beta}2, interacted with the first PDZ domain of NHERF-1, forming a multiprotein signaling complex. Given the presence of NHERF-2 in DVR and the role of Ca2+ in the function of its constituent cells, we initiated experiments to determine whether either TRPC4 or TRPC5 is expressed in DVR and, if so, whether a specific association with NHERF-2 exists.

Our studies demonstrate that DVR express TRPC4 and NHERF-2 message and protein. Using hand-dissected DVR and RT-PCR, mRNA for both proteins was identified (Fig. 1). Using RT-PCR, we did not detect TRPC5 in isolated DVR. We detected TRPC5 using Western immunoblot analysis of medullary lysates (Fig. 2), so it remains possible that TRPC5 is expressed in another structure within the renal medulla. Detailed confocal microscopic studies indicated that both TRPC4 and NHERF-2 colocalize in some cells of the DVR wall (Figs. 47). The physical interaction between these proteins was confirmed by performing immunoprecipitation experiments (Fig. 8). Antibody directed against NHERF-2 immunoprecipitates TRPC4 from lysates of the renal medulla. Using an antibody against NHERF-1, we failed to detect either TRPC4 or TRPC5 in immunoprecipitate derived from lysates of the cortex or medulla. Taken together, these findings suggest that TRPC4 is present in the cells of the DVR and associates with NHERF-2. To our knowledge, this represents the first in vivo demonstration of a TRPC4-NHERF-2 interaction. Through its PDZ binding domain, TRPC5 also has the potential to interact with NHERF-2. We were unable, however, to detect TRPC5 message using RT-PCR or protein using immunochemistry, which implies that such an association does not exist in the cells of the DVR. We recognize, however, that the quality of the available TRPC5 antibody may have limited detection and may require reevaluation when better reagents are available.

The function of TRPC4 and the significance of its binding to NHERF-2 are unknown at present. TRPC4 and TRPC5 of the TRPC subfamily have been proposed to function as nonselective cation channels (5, 9, 11, 21, 25). Freichel et al. (5) reported their studies in TRPC4–/– mice. A striking observation in those experiments was the marked attenuation of agonist-induced increases in intracellular Ca2+ in endothelial cells deficient in TRPC4. Freichel et al. also demonstrated impaired agonist-induced vasorelaxation in preconstricted aortic rings from TRPC4-null mice. They interpreted their results as showing that TRPC4 functions as a store-operated Ca2+ channel that mediates capacitive Ca2+ entry, at least in endothelia of a large conduit vessel. While these data are intriguing, we think it is important to emphasize that it has not firmly been established that TRPC4 is the only store-operated Ca2+ channel or that all endothelial cells, especially microvascular endothelial cells, use similar mechanisms. Recently, Sansom et al. (25) reported that TRPC4 forms store-operated Ca2+ channels in mouse kidney mesangial cells. As shown in Fig. 4B, we also observed immunological evidence of TRPC4 in glomeruli, where it might also be associated with NHERF-2.

Study of DVR is difficult because this structure is inaccessible in the intact kidney. A major advance has been the development of ex vivo methods that permit delineation of DVR responses to hormones, activation of specific ion channels, and modulation of cytoplasmic Ca2+ concentrations in both pericytes and endothelia. Investigations have repeatedly demonstrated that activation of Ca2+-mediated signaling pathways plays a vital role in the regulation of vasoactivity and the release of NO (1215, 18, 2729). Those functional studies have not permitted delineation of the importance of interactions between specific signaling proteins, receptors, and ion channels. The present study indicates that TRPC4 and NHERF-2 associate with one another in DVR, which provides a rationale for investigations of the physiological role of these proteins in the regulation of medullary blood flow.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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These studies were supported by National Institutes of Health Grants DK-42495, HL-62220/DK-67621, and DK-68492 (to T. L. Pallone), DK-32839 (to J. B. Wade), and DK-55881 (to E. J. Weinman) and by a grant from the Research Service of the Department of Veterans Affairs (to E. J. Weinman).


    ACKNOWLEDGMENTS
 
We acknowledge the contributions of Deborah Steplock and Jie Liu to these experiments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: E. J. Weinman, Division of Nephrology, Department of Medicine, Univ. Maryland School of Medicine, N3W143, UMH, 22 S. Greene St., Baltimore, MD 21201 (E-mail: eweinman{at}medicine.umaryland.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Breton S, Wiederhold T, Marshansky V, Nsumu NN, Ramesh V, and Brown D. The B1 subunit of the H+ATPase is a PDZ domain-binding protein: colocalization with NHE-RF in renal B-intercalated cells. J Biol Chem 275: 18219–18224, 2000.[Abstract/Free Full Text]

2. Brezis M, Heyman SN, and Epstein FH. Determinants of intrarenal oxygenation. II. Hemodynamic effects. Am J Physiol Renal Fluid Electrolyte Physiol 267: F1063–F1068, 1994.[Abstract/Free Full Text]

3. Brezis M and Rosen S. Hypoxia of the renal medulla: its implications for disease. N Engl J Med 332: 647–655, 1995.[Free Full Text]

4. Cowley AW. Role of the renal medulla in arterial blood pressure regulation. Am J Physiol Regul Integr Comp Physiol 273: R1–R15, 1997.[Abstract/Free Full Text]

5. Freichel M, Suh SH, Pfeifer A, Schweig U, Trost C, Weissgerber P, Biel M, Philipp S, Freise D, Droogmans G, Hofmann F, Flockerzi V, and Nilius B. Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4–/– mice. Nat Cell Biol 3: 121–127, 2001.[CrossRef][ISI][Medline]

6. Lemley KV and Kriz W. Cycles and separations: the histotopography of the urinary concentrating process. Kidney Int 31: 538–548, 1987.[ISI][Medline]

7. Mattson DL. Importance of the renal medullary circulation in the control of sodium excretion and blood pressure. Am J Physiol Regul Integr Comp Physiol 284: R13–R27, 2003.[Abstract/Free Full Text]

8. Mery L, Strauss B, Dufour JF, Krause KH, and Hoth M. The PDZ-interacting domain of TRPC4 controls its localization and surface expression in HEK293 cells. J Cell Sci 115: 3497–3508, 2002.[Abstract/Free Full Text]

9. Minke B and Cook B. TRP channel proteins and signal transduction. Physiol Rev 82: 429–472, 2002.[Abstract/Free Full Text]

10. Montell C, Birnbaumer L, and Flockerzi V. The TRP channels, a remarkably functional family. Cell 108: 595–598, 2002.[CrossRef][ISI][Medline]

11. Pak WL and Leung HT. Genetic approaches to visual transduction in Drosophila melanogaster. Receptors Channels 9: 149–167, 2003.[CrossRef][ISI][Medline]

12. Pallone TL, Edwards AE, Turner MR, and Jamison RL. Countercurrent exchange in the renal medulla. Am J Physiol Regul Integr Comp Physiol 284: R1153–R1175, 2003.[Abstract/Free Full Text]

13. Pallone TL and Huang JM. Control of descending vasa recta pericyte membrane potential by angiotensin II. Am J Physiol Renal Physiol 282: F1064–F1074, 2002.[Abstract/Free Full Text]

14. Pallone TL and Silldorff EP. Pericyte regulation of renal medullary blood flow. Exp Nephrol 9: 165–170, 2001.[CrossRef][ISI][Medline]

15. Pallone TL, Zhang Z, and Rhinehart K. Physiology of the renal medullary microcirculation. Am J Physiol Renal Physiol 284: F253–F266, 2003.[Abstract/Free Full Text]

16. Philipp S, Trost C, Warnat J, Rautmann J, Himmerkus N, Schroth G, Kretz O, Nastainczyk W, Cavalie A, Hoth M, and Flockerzi V. TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells. J Biol Chem 275: 23965–23972, 2000.[Abstract/Free Full Text]

17. Rhinehart K, Handelsman CA, Silldorff EP, and Pallone TL. Angiotensin II AT2 receptor modulates descending vasa recta endothelial Ca2+ signaling. Am J Physiol Heart Circ Physiol 284: H779–H789, 2003.[Abstract/Free Full Text]

18. Rhinehart K and Pallone TL. Nitric oxide generation by isolated descending vasa recta. Am J Physiol Heart Circ Physiol 281: H316–H324, 2001.[Abstract/Free Full Text]

19. Shenolikar S and Weinman EJ. NHERF: targeting and trafficking membrane proteins. Am J Physiol Renal Physiol 280: F389–F395, 2001.[Abstract/Free Full Text]

20. Tang Y, Tang J, Chen Z, Trost C, Flockerzi V, Li M, Ramesh V, and Zhu MX. Association of mammalian trp4 and phospholipase C isozymes with a PDZ domain-containing protein, NHERF. J Biol Chem 275: 37559–37564, 2000.[Abstract/Free Full Text]

21. Vennekens R, Voets T, Bindels RJ, Droogmans G, and Nilius B. Current understanding of mammalian TRP homologues. Cell Calcium 31: 253–264, 2002.[CrossRef][ISI][Medline]

22. Voltz JW, Weinman EJ, and Shenolikar S. Expanding the role of NHERF, a PDZ domain containing adaptor to growth regulation. Oncogene 20: 6309–6314, 2001.[CrossRef][ISI][Medline]

23. Wade JB, Liu J, Coleman RA, Cunningham R, Steplock D, Lee-Kwon W, Pallone TL, Shenolikar S, and Weinman EJ. Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse. Am J Physiol Cell Physiol 285: C1494–C1504, 2003.[Abstract/Free Full Text]

24. Wade JB, Welling P, Donowitz M, Shenolikar S, and Weinman EJ. Differential renal distribution of NHERF isoforms and their co-localization with NHE3, ezrin, and ROMK. Am J Physiol Cell Physiol 280: C192–C198, 2001.[Abstract/Free Full Text]

25. Wang X, Pluznick JL, Wei P, Padanilam BJ, and Sansom SC. TRPC4 forms store-operated Ca2+ channels in mouse mesangial cells. Am J Physiol Cell Physiol 287: C357–C364, 2004.[Abstract/Free Full Text]

26. Weinman EJ, Minkoff C, and Shenolikar S. Signal-Complex regulation of renal transport proteins: NHERF and the regulation of NHE3 by PKA. Am J Physiol Renal Physiol 279: F393–F399, 2000.[Abstract/Free Full Text]

27. Zhang Z and Pallone TL. Response of descending vasa recta endothelia to luminal pressure. Am J Physiol Renal Physiol 287: F535–F542, 2004.[Abstract/Free Full Text]

28. Zhang Z, Rhinehart K, Kwon W, Weinman E, and Pallone TL. ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species. Am J Physiol Heart Circ Physiol 287: H773–H781, 2004.[Abstract/Free Full Text]

29. Zhang Z, Rhinehart K, and Pallone TL. Membrane potential controls calcium entry into descending vasa recta pericytes. Am J Physiol Regul Integr Comp Physiol 283: R949–R957, 2002.[Abstract/Free Full Text]