INTRODUCTION

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THE KIDNEY PLAYS A KEY ROLE in Ca²⁺ (and Mg²⁺) homeostasis by adjusting the tubular reabsorption of these divalent cations from the glomerular filtrate. Parathyroid hormone (PTH) and calcitonin, as well as vitamin D (23, 34), play important roles in regulating divalent mineral excretion. In the absence of these calciotropic factors, however, the steep relationship between plasma and urinary calcium is preserved in both rat (22) and humans (4). This suggests that a "fourth" calciotropic factor is involved in regulating renal Ca²⁺ excretion (19).

Recent evidence indicates that extracellular Ca²⁺ itself, interacting with the calcium-sensing receptor (CaR), provides this regulatory function (19). Transcripts for the extracellular Ca²⁺/polyvalent cation-sensing G protein-coupled receptor are expressed in rat (32) and bovine (11) kidney, particularly in proximal and distal nephron segments involved in divalent mineral reabsorption. In addition, there is abnormal renal Ca²⁺ sensing in familial hypocalciuric hypercalcemia (FHH), an inherited disorder with mutations in the CaR gene resulting in receptors with severely lowered or abolished responses to extracellular Ca²⁺ (28). Individuals with FHH exhibit little-to-no rise in urinary Ca²⁺ excretion with increasing plasma calcium; however, administration of furosemide enhances renal Ca²⁺ excretion in these individuals with FHH, suggesting that Ca²⁺ reabsorption is abnormally high in the thick ascending limb (TAL) (4).

A number of other studies have also supported a role for the renal CaR in modulating kidney function (10, 21). For example, increases in extracellular Ca²⁺ (or Mg²⁺) concentrations directly modulate mineral ion transport in the loop of Henle and distal convoluted tubule (DCT) (9, 24, 30, 31). More recently, direct roles for RaKCaR in modulating renal function have been suggested for two rat nephron segments. In the rat TAL, extracellular Ca²⁺, presumably via the Ca²⁺-sensing receptor, reversibly inhibits apical K⁺ channel activity (39, 40), an effect which would both reduce NaCl absorption, and consequently urinary concentrating power, and divalent mineral reabsorption (20). In addition, CaR protein has been identified by immunohistochemistry at the apical border of both human and rat terminal collecting ducts, where increases in perfusate Ca²⁺ concentrations reversibly reduce arginine vasopressin (AVP)-stimulated water reabsorption (36).

In the present study, we investigate the renal segmental distribution and cellular localization of CaR protein. Some of the potential roles of the CaR in these nephron segments are discussed.

	MATERIALS AND METHODS

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Affinity purification of anti-CaR antibody and immunoabsorption. The polyclonal rabbit anti-bovine CaR (antiserum 4641; a gift from NPS Pharmaceuticals, Salt Lake City, UT) used in the present study has been characterized previously (12, 36). The antibody was raised to residues 215-237 within the extracellular domain of the bovine CaR, which are identical to residues 214-236 of the rat CaR. On Western blots of rat kidney protein, the anti-CaR antibody recognizes bands of 135 and 200-235 kDa. Purification of anti-CaR antiserum was performed as described previously (42). Briefly, 3 ml carboxy-activated support (Bio-Rad Laboratories) was washed with 25 ml dry DMSO using vacuum filtration on a glass fitted funnel. One milligram of the CaR peptide was dissolved in 20 ml dry DMSO and added to the washed, activated support. A quantity of 100 µl dry triethylamine was then added to the suspension and incubated overnight at room temperature on a rocking shaker. A volume of 500 µl ethanolamine was subsequently added to the suspension to block remaining activated carboxylates. The support was then washed three times with 20 ml DMSO, several times with 1 N acetic acid, and finally with distilled water. The resulting support was poured into a chromatography column and equilibrated with 5× PBS. A quantity of 500 µl antiserum (diluted 1:1 with 10× PBS) was added to the column and incubated at 4°C for overnight. The column was then washed five times with 5× PBS. Finally, purified antibody was eluted from the column with 3 ml of 100 mM sodium citrate, pH 2.5, and was neutralized immediately with 1.5 ml of 1 M Tris hydrochloride, pH 8.5. The purified antibody was dialyzed against 1× PBS at 4°C for 24 h and concentrated to a final volume of 500 µl. Immunoabsorbed serum was generated by overnight incubation of anti-CaR antibody with CaR peptide support at 4°C.

Immunofluorescence. Localization of RaKCaR protein along the nephron was performed using antibodies that recognize specific antigens in certain nephron segment. DCTs were identified using a rabbit polyclonal antiserum raised against the thiazide-sensitive Na⁺-Cl cotransporter (anti-rTSC, 1:1,000; Ref. 27). Intercalated cells in cortical collecting ducts (CCDs) were identified using a polyclonal antibody raised against the vacuolar type H⁺-ATPase (at 1:400 dilution; a gift from Dr. Dennis Brown; Ref. 7). Type A intercalated cells of the collecting ducts were identified using anti-band 3 anion exchanger antibody (AE1, at 1:500 dilution; a gift from Dr. S. Alper; see Refs. 6 and 38). Finally, macula densa cells were identified with a mouse monoclonal antibody specific for the brain type nitric oxide synthase I (NOS-B1, at 1:125 dilution; Sigma; see Ref. 26), which does not exhibit any cross reactivity with the inducible and endothelial isoforms of NOS (NOS II and NOS III, respectively).

Male Sprague-Dawley rats (100-125 g, Harlan) were anesthetized, and kidneys were perfusion fixed via the descending aorta with 4% paraformaldehyde, followed by 750 or 1,250 mosmol/kg PBS/sucrose solution. RaKCaR fluorescence was generally optimal when the tissue was perfused with a solution hypertonic with respect to plasma osmolality. Sagittal sections were cryoprotected overnight at 4°C in 30% sucrose in PBS. Tissue was embedded in OCT (Miles, Elkhart, IN) and frozen in isopentane cooled on dry ice. Three-micrometer frozen sections were cut with a Leica CM3050 cryostat and thaw mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). Tissue cryosections were antigen retrieved using a citrate-containing buffer (CITRA; BioGenex, San Ramon, CA), followed by treatment with 1% SDS in PBS for 5 min to expose antigenic sites (8). After SDS treatment, slides were rinsed three times with PBS, incubated 30 min with 1% BSA/PBS followed by two 5-min washes in "high-salt" PBS + 2.8% wt/vol NaCl, then two 5-min washes in PBS. Slides were then incubated with 4% Seablock (SeaRun Holdings, Arundel, ME) for 1 h at room temperature followed by overnight incubation with primary affinity-purified anti-CaR polyclonal antibody (at 1:100-1:1,000 dilution) and, where indicated, a second primary against Na⁺-Cl cotransporter (rTSC1), H⁺-ATPase, anion exchanger type 1 (AE1), or nitric oxide synthase I (NOS-B1). To assess nonspecific staining, control experiments were performed by incubating the slides without primary antibody or with primary antibody blocked by preincubation with an excess of peptide for 1 h at room temperature. Secondary antibodies were diluted with 1% BSA/PBS and applied to sections for 1 h at room temperature at the following dilutions: Texas Red-conjugated anti-rabbit IgG, 1:200 (Jackson Immunochemicals, West Grove, PA); Rhodamine Red-X-conjugated Fab fragment anti-rabbit IgG, 1:200 (Jackson Immunochemicals) diluted in PBS pH 8.2; and FITC-conjugated anti-rabbit IgG, 1:200 (Vector Laboratories, Burlingame, CA). When staining with two primary antibodies, the first fluorescence-labeled secondary antibody was Rhodamine Red-X-conjugated Fab fragment anti-rabbit IgG, and the second secondary antibody was FITC-conjugated anti-rabbit IgG. To ensure saturation of all antigenic sites on the first primary antibody, after incubation with the Rhodamine Red-X-conjugated Fab fragment anti-rabbit IgG, sections were incubated with AffiniPure Fab fragment goat anti-rabbit (1:20; Jackson Immunochemicals) for 1 h at room temperature before continuing with the second primary antibody. When using a single primary and both secondary antibodies, we find no significant FITC fluorescence using this dual staining method. Sections were examined with a Zeiss LSM410 laser-scanning confocal microscope or a Nikon Eclipse 800 Research microscope, and fluorescence photomicrographs were taken using Kodak color Elite 400 and T-Max films for color and black and white pictures, respectively.

	RESULTS

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Immune serum that was affinity purified using the CaSR peptide showed intense immunofluorescence of tubule profiles in the rat cortex (Fig. 1, A and B). No significant fluorescence was observed using in any of the structures in the cortex (or other regions of the kidney) with peptide-preabsorbed serum (Fig. 1, C and D) or with preimmune serum (Fig. 1, E and F). Western blots of kidney protein using this antibody have been shown previously to recognize bands of 135 and 200-235 kDa, with the latter probably representing receptor dimers (36). Thus the polyclonal anti-CaSR antibody specifically recognizes CaSR protein in rat kidney.

View larger version (167K): [in this window] [in a new window]   View larger version (108K): [in this window] [in a new window]   View larger version (173K): [in this window] [in a new window]   View larger version (59K): [in this window] [in a new window]   View larger version (176K): [in this window] [in a new window]   View larger version (84K): [in this window] [in a new window]   Fig. 1.   Low-magnification (×88) images in rat kidney cortex. A and B: phase-contrast (A) and CaR-specific fluorescence (B) in kidney cortex. C and D: phase-contrast image (C) and fluorescence with peptide preabsorbed immune serum (D). E and F: phase-contrast image (E) and fluorescence with preimmune serum (F).

The general distributions of CaR-specific fluorescence in the rat cortex and outer medulla are shown in Fig. 2, A-D. Many tubule profiles exhibited staining in the cortex (Fig. 2, A and B) as well as in both outer (Fig. 2C) and inner (Fig. 2D) stripes of the outer medulla. The most intense staining was observed in cortical TAL segments (Fig. 2A and in the medullary ray in Fig. 2B). Medullary TAL segments in both outer (Fig. 2C) and inner (Fig. 2D) stripes of outer medulla were also stained. CaR-specific fluorescence was also seen at the apical border of many proximal tubule profiles (Fig. 2, A and B). Faint fluorescence was also detected at the apical border of terminal collecting ducts (not shown) in a pattern consistent with the immunohistochemical staining published previously (36). No significant fluorescence was detected in glomeruli (Fig. 2, A and B) or blood vessels (not shown) with affinity-purified immune serum (Fig. 1).

View larger version (213K): [in this window] [in a new window]   Fig. 2.   Low-magnification (×85) views showing CaR-specific fluorescence in outer cortex (A), midcortex (B), and outer (C) and inner (D) stripes of outer medulla (OM) of rat kidney; g, glomeruli; open arrow, brightly fluorescent thick ascending limb (TAL) profiles; double-headed arrows, staining at the apical border of proximal tubule. IM, inner medulla.

In proximal tubule, Ca²⁺ receptor staining was greatest in S1 segments immediately following the glomerulus (Figs. 3A and 4C). Comparing the high-magnification (×1,000) differential-interference contrast (Fig. 4A) and rhodamine fluorescence (Fig. 4B) images of the same proximal tubule, it is clear that the Ca²⁺-sensing receptor is specifically localized to the base of the brush border of S1 segments (Fig. 4, A and B). Apical CaR-specific fluorescence intensity diminished along the proximal tubule from S1 to S3 segments. In proximal S2 segments, CaR staining was diminished compared with that in early S1 segments [compare Fig. 3B (S1) with Fig. 3C (S2); Fig. 4, B and C (S1), with Fig. 4D (S2); and in Fig. 4D (S1 and S2)]. In some S2 segments with little or no apical Ca²⁺-sensing receptor fluorescence, intense staining was seen in large vesicle-like structures (Fig. 4E) reminiscent of those observed in certain circumstances with antibody against the Na⁺- cotransporter, NaPi2 (25). In contrast, CaR-specific fluorescence was absent in S3 proximal straight tubule profiles in outer stripe of outer medulla (Fig. 2C).

View larger version (133K): [in this window] [in a new window]   Fig. 3.   High-magnification images of CaR-specific fluorescence in rat kidney cortex. A: outer cortex (×340) showing apical fluorescence in an initial S1 proximal tubule (arrow) exiting from glomerulus labeled (G). B: higher magnification (×510) of proximal S1 segments shows apical fluorescence (arrow). C: proximal S2 segments (×510) show less intense CaR-specific fluorescence at the apical border (arrow) than in S1 (compare proximal tubule profiles in A and B with those in C). D: a cortical medullary ray (×340) showing both brightly fluorescent TAL segments (open arrows) as well as significant signal in certain cells (solid arrows) in cortical collecting duct (CCD, labeled as "C"). These latter cells in the CCD were identified as type A intercalated cells. E: apical CaR-specific staining in a proximal tubule (PT) next to a distal convoluted tubule (DCT). Note the basolateral signal in DCT as well as the bright punctate fluorescence (arrow) at the apical surface suggestive of intracellular subapical vesicles.

View larger version (K): [in this window] [in a new window]   Fig. 4.   CaR-specific fluorescence (rhodamine) in proximal tubule. A and B: a proximal S1 segment at high magnification (×870) shown using differential-interference contrast (A) or CaR-specific rhodamine fluorescence (B). In A and B, note the fluorescence is primarily at the base of the microvilli (arrow). C: a high-magnification (×520) image showing CaR-specific fluorescence at the apical border (arrow) of a proximal S1 segment emerging from a glomerulus. D: CaR-specific fluorescence (×520) in proximal S1 (open arrow) and proximal S2 (solid arrow) tubule profiles; note that the fluorescence intensity is less in S2 than in S1 segments. E: high-magnification (×870) image showing CaR-specific fluorescence in a vesicle-like structure (arrow) in a late S2 proximal tubule.

CaR-specific fluorescence was detected along the entire length of the TAL [cortical TAL (Fig. 2B) > medullary TAL (Fig. 2, C and D); TAL profiles are shown in the medullary ray in Fig. 3D]. In confocal images shown in Fig. 5, A and B, Ca²⁺-sensing receptor fluorescence is restricted to the basolateral border in cortical TAL segments in a pattern consistent with the deep basolateral membrane infoldings. Medullary TAL segments exhibited a similar CaR-specific fluorescence pattern (not shown). There was considerable cell-to-cell variability in CaR-specific fluorescence in both cortical and medullary TAL segments (e.g., see Fig. 5, A and B).

View larger version (54K): [in this window] [in a new window]   Fig. 5.   Confocal images of cortical TAL segments (A and B) and a DCT (C and D) at a magnification of ×800. Note the basolateral pattern of CaR-specific fluorescence in cortical TAL segments (arrows, A and B). DCT is costained with anti-CaR antibody (C) and anti-TSC1 (Na+-Cl cotransporter) antibody (D). Note that CaR-specific fluorescence is basolateral (arrow, C), whereas that for the cotransporter (arrow, D) is apical.

In the region of the cortical TAL near glomeruli, we occasionally observed large cells with the morphological appearance of macula densa cells (Fig. 6A) that also showed intense basolateral staining for the Ca²⁺-sensing receptor (Fig. 6B). We confirmed the presence, as well as the basolateral localization, of the Ca²⁺-sensing receptor protein in macula densa cells (rhodamine fluorescence, Fig. 6D) by costaining the same section with anti-NOS-B1 antibody (FITC fluorescence, Fig. 6C).

View larger version (K): [in this window] [in a new window]   Fig. 6.   A-D: images of macula densa. Phase-contrast (A) and CaR-specific rhodamine fluorescence (B) images of the cortical TAL at the macula densa (×310). Large macula densa cells are shown by the solid arrows in A and the open arrow in B next to glomerulus (labeled "G"). Note the basolateral localization of CaR fluorescence in macula densa cells (B, solid arrow, TAL cells exhibit CaR-specific fluorescence). The same macula densa is shown in C and D stained with anti-brain type nitric oxide synthase I (C; FITC fluorescence) and anti-CaR (D; rhodamine fluorescence) antibodies. E: TAL (T) with associated DCT profiles (D) showing CaR-specific fluorescence (×310); note the decrease in fluorescence intensity at the transition from TAL to DCT (solid arrow). F and G: DCT costained for CaR (rhodamine fluorescence) and the apical thiazide-sensitive Na+-Cl cotransporter, rTSC1 (FITC fluorescence; F at ×310 and G at ×470). Note the basolateral localization of the CaR fluorescence. Arrow in F, a rTSC1-negative cell that exhibits intense CaR-specific fluorescence. These cells were identified as type A intercalated cells. Some DCT tubules identified by rTSC1-specific apical fluorescence (lumen "2", arrow in G) did not exhibit CaR-specific fluorescence. These latter tubules are likely late DCT or in the DCT-CNT transition zone. H: high-magnification (×780) image of a DCT costained with anti-CaR (rhodamine fluorescence) and anti-H+-ATPase (FITC fluorescence) antibodies. Open arrow, type A intercalated cell with apical H+-ATPase and basolateral CaR-specific fluorescence signals; solid arrow, subapical vesicle-like structures in a DCT cell.

At the transition of the cortical TAL and DCT, Ca²⁺-sensing receptor fluorescence became abruptly less intense (Fig. 6E). This reduced, but significant, CaR-specific staining continued following the TAL-DCT transition in groups of tubule profiles (Fig. 6E). As shown in Fig. 6F, this latter staining for the Ca²⁺-sensing receptor (rhodamine fluorescence) was basolateral and in tubule profiles exhibiting apical fluorescence with anti-rTSC1 antibody (FITC fluorescence), indicating that these were DCT cells (27). Confocal images of DCT profiles costained with anti-rTSC1 (Fig. 5C) and anti-CaR (Fig. 5D) antibodies clearly show the localization of these proteins in apical and basolateral borders, respectively. In general, anti-Ca²⁺-sensing receptor fluorescence (rhodamine) diminished along the distal tubule (Fig. 6E), and in some rTSC1-positive tubule profiles (apical FITC stained) basolateral Ca²⁺-sensing receptor staining was absent (compare the two tubule profiles in Fig. 6G). The rTSC1-positive, CaR-negative tubule profiles (e.g., lumen "2" Fig. 6G) were smaller in diameter than the rTSC1-positive, CaR-positive tubule profiles (e.g., lumen "1" in Fig. 6G), suggesting that the former were DCT-connecting segment (CNT) transition zones or early CNT (27). In addition, intense Ca²⁺-sensing receptor staining in the DCT was observed in a minority cell population (Fig. 6F) that was rTSC1 negative. These latter cells were identified as intercalated cells by their apical fluorescence for H⁺-ATPase (green FITC fluorescence, Fig. 6H). Finally, intense Ca²⁺-sensing receptor staining was often observed in a punctate pattern at the apical border in some DCT cells, suggesting expression in intracellular vesicles (Figs. 3E and 6H).

In the CCD, Ca²⁺-sensing receptor fluorescence was seen in a minority cell population (Fig. 3D). The pattern of cellular fluorescence varied considerably in these cells from staining of the entire cytosol (majority of cells) to staining at the apical or basolateral surface (minority of positive cells). Costaining of sections for both CaR (rhodamine) and H⁺-ATPase or AE1 (FITC) indicated that the CaR-positive cells were type A intercalated cells (Fig. 7). Shown in Fig. 7A, we find the typical staining pattern for apical vacuolar H⁺-ATPase (rhodamine) and basolateral Cl- cotransporter (AE1FITC) described for type A intercalated cells in the CCD (1). In the early collecting duct, CaR-positive cells had a diffuse cytosolic pattern and did not express AE1 staining. These cells are typical of intercalated cells with a diffuse cytosolic H⁺-ATPase staining pattern (1, 35). Figure 7, D-F, shows the same section costained with anti-CaR (rhodamine fluorescence) and anti-H⁺-ATPase (FITC fluorescence) antibodies and exposed using the dual (Fig. 7D), rhodamine (Fig. 7E), and FITC (Fig. 7F) filters. Cells with both diffuse and apical H⁺-ATPase staining are seen to express CaR in a similar pattern. Not all type A intercalated cells were CaR positive. Five type A intercalated cells are shown at high-magnification (×1,000) in a typical CCD stained for both CaR (rhodamine) and H⁺-ATPase (FITC) using the dual filter (Fig. 7G) or the rhodamine filter (Fig. 7H). It is evident that CaR colocalizes to only three (Fig. 7H) of the five H⁺-ATPase-positive cells.

View larger version (K): [in this window] [in a new window]   Fig. 7.   CaR-specific fluorescence in CCD. A: a section showing a CCD costained with anti-AE1 [Cl/ exchanger; FITC (yellow-green fluorescence)] and anti-H+-ATPase (rhodamine fluorescence) antibodies; , four type A intercalated cells with apical H+-ATPase and basal Cl/ exchanger staining. Arrows, three intercalated cells exhibiting diffuse staining for H+-ATPase. B and C: same section of a CCD in the outer cortex costained with anti-AE1 (FITC fluorescence) and anti-CaR (rhodamine fluorescence) antibodies and imaged using the dual (B) or rhodamine (C) filter. Note that the three lightly CaR-staining cells (arrows in C) do not stain with anti-AE1 antibody. D-F: same section showing a CCD (labeled "C") costained with anti-CaR (rhodamine) and anti-H+-ATPase (FITC) antibodies and imaged with a dual (D), rhodamine (E), or FITC (F) filter; "T," cortical TAL profiles; arrows in D-F, four intercalated cells exhibiting diffuse staining for H+-ATPase; , two type A intercalated cells with apical H+-ATPase staining. Note that H+-ATPase and CaR colocalize in these cells. G and H: high magnification (×780) of a CCD costained for CaR (rhodamine fluorescence) and H+-ATPase (FITC fluorescence) and imaged using the dual (G) or FITC (H) filter. Five type A intercalated cells are shown staining for H+-ATPase (arrows, G), but only three cells exhibit CaR-specific fluorescence (open arrows, H).

	DISCUSSION

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Transcripts for the G protein-coupled, Ca²⁺/polyvalent cation-sensing receptor cloned from rat kidney (32) are present in glomeruli as well as in many nephron segments from proximal tubule to inner medullary collecting duct (IMCD) (33). In an earlier study in rat kidney, Sands and co-workers (36) showed that CaR protein was expressed at the apical border of cells in the terminal IMCD and showed that purified subapical endosomes contained both CaR and the water channel, aquaporin-2. In the present study, we have extended the receptor protein localization in rat kidney to include both cortex and outer medulla. The CaR protein was shown to be present in the proximal tubule, medullary and cortical TAL segments, macula densa, DCT, and type A intercalated cells in the distal tubule and CCD. Interestingly, the polarity of CaR protein expression varied along the rat nephron, being apical in proximal tubule (and IMCD, Ref. 36) but basolateral in medullary TAL, cortical TAL, and DCT. A variable but mainly cytosolic pattern of CaR staining was seen in intercalated cells in the CCD. Thus this receptor can apparently be trafficked either to the apical or to the basolateral membrane depending on tubule cell type. In general, the CaR protein localization shown here agreed with that for CaR transcripts in rat kidney (33), except for the lack of immunofluorescence signal in glomeruli. Apparently, CaR protein expression in glomeruli is either absent or below the detection limits of fluorescence using this polyclonal anti-CaR antibody.

We localized CaR protein to the luminal border of both proximal convoluted and straight tubule cells. Potential roles for the CaR in proximal tubule function have been suggested and include modulation of 1-hydroxylation of 25-hydroxyvitamin D₃ and PTH-stimulated second messenger production (10, 19). The apical localization of the CaR in proximal tubule also suggests potential roles for luminal divalent minerals and/or polyvalent cations in regulating solute reabsorption processes including bicarbonate or phosphate. Studies examining some of these possibilities are currently underway.

Not surprisingly, the most intense fluorescence signal for the CaR was in cortical TAL segments, which are known to reabsorb divalent minerals in a regulated manner (18, 30). Given the intense fluorescence in cortical TAL cells, however, we cannot exclude that some of the CaR fluorescence was intracellular, either representing receptor in the process of being synthesized and/or in intracellular compartments such as mitochondria. The localization of the Ca²⁺-sensing receptor at the basolateral border of TAL cells is consistent with roles for the receptor in sensing plasma Ca²⁺/Mg²⁺ and in potentially providing a negative feedback response to divalent minerals being absorbed via the paracellular pathway.

Exposing mouse isolated TAL tubules to increasing Ca²⁺ concentrations has been shown to reduce AVP-stimulated cAMP accumulation (37). Since this decrease in hormone-dependent cAMP accumulation was pertussis toxin sensitive, it was suggested that high extracellular Ca²⁺ activated the inhibitory G protein, G_i. In a recent study, activation of G_i by the extracellular Ca²⁺/polyvalent cation-sensing receptor was shown in MDCK cells that exhibit "distal tubule"-like characteristics (3). In this latter study, CaR activated G_i-2 and G_i-3 but not G_i-1. The CaR-dependent activation of G_i could be indirect in the TAL. In rat TAL, CaR activation stimulates phospholipase A₂ and the release of arachidonic acid (40), and arachidonic acid has been shown to reduce hormone-stimulated cAMP production, in a pertussis toxin-sensitive manner, (17).

In vivo perfusion studies in rat kidney have demonstrated that increasing basolateral Ca²⁺ and Mg²⁺ concentrations reduces NaCl and divalent mineral reabsorption by the TAL (16, 29-31). Thus the localization of the CaR in TAL shown in the present report can provide the sensing mechanism accounting for regulation of TAL transport function by extracellular divalent minerals and possibly other polyvalent cations like aminoglycoside antibiotics. A model described recently for the involvement of CaR in regulating ion transport by the TAL (39, 40) suggests that CaR inhibits the apical 70-pS K⁺ channel and the Na⁺-K⁺-2Cl cotransport activity, therefore reducing NaCl absorption via a cytochrome P-450 metabolism pathway (2, 5, 16, 18, 20, 41). Furthermore, extracellular Ca²⁺-mediated inhibition of TAL NaCl absorption would reduce countercurrent multiplication, and therefore lower urinary concentrating ability, and allow for excretion of divalent minerals in a more dilute urine. Thus these effects of CaR activation in the TAL can account, at least in part, for the CaR-dependent relationship between plasma divalent mineral concentrations and Ca²⁺/Mg²⁺ urinary excretion (see Introduction; also, Refs. 4 and 22).

CaR protein was expressed at the basolateral border of macula densa cells (Fig. 5, A-D), where receptor sensing of variations in extracellular Ca²⁺ could play a role in modulating glomerulotubular feedback responses. CaR protein was also detected at the basolateral border in DCT identified by costaining with antibody to the thiazide-sensitive Na⁺-Cl cotransporter. Active Ca²⁺ reabsorption by the DCT is regulated by calciotropic factors (18) and is inversely related to Na⁺ reabsorption (15). Although roles for the CaR in DCT function have not been specifically identified, increases in extracellular Ca²⁺ have been shown to modulate the calcium binding protein calbindin D28K gene expression in primary chick kidney cells (13). Whether extracellular divalent minerals modulate Ca²⁺/Mg²⁺ transport in DCT via the CaR remains to be examined. In some DCT profiles, CaR fluorescence also showed a punctate apical staining pattern. Recently, the thiazide-sensitive Na⁺-Cl cotransporter protein has been identified, not only on apical membranes of DCT segments, but also in subapical vesicles (27). Since inhibition of the Na⁺-Cl cotransporter modulates Ca²⁺ reabsorption by the DCT (14, 18), it is possible that luminal Ca²⁺ might modulate Na⁺-Cl cotransporter trafficking to, or from, the apical membrane via a mechanism analogous to that proposed for Ca²⁺-CaR modulation of water transport in the IMCD (36).

Finally, we demonstrated expression of CaR protein in type A intercalated cells in DCT and CCD, suggesting a potential role for extracellular divalent minerals in regulating proton secretion. Since urine pH can modulate divalent mineral solubility, it seems reasonable to suggest that extracellular Ca²⁺-mediated regulation of urine acidification could play an important role in reducing the risk of stone formation or nephrocalcinosis.