Localization of Receptors for 1,25-Dihydroxyvitamin D3 along the Rat Nephron DIRECT EVIDENCE FOR PRESENCE OF THE RECEPTORS IN BOTH PROXIMAL AND DISTAL NEPHRON*

We recently demonstrated that 25-hydroxyvitamin D3-24-hydroxylase, which can be further induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is localized exclusively in the proximal nephron of the vitamin D- replete rat kidney. These data and the proposed mode of action of 1,25(OH)& predict the presence of a receptor for 1,25(OH)&3 in the proximal nephron. However, autoradiographic studies failed to detect significant nuclear uptake of 1,25(0H)~D3[~H]D~ in the proximal nephron. To localize and characterize receptors for 1,25-(OH)2D3 along the nephron, preparations of both micro- dissected defined nephron segments and isolated tubules from the whole kidney of vitamin D-deficient rats were incubated with 1,25(OH)2[3H]D3, and specific up- take of the sterol was assessed in the cytosol preparation of tubular cells sonicated in a hypertonic solution. The preparation of isolated tubules incubated for 1 h at 37 “C accumulated 1,25(OH)2[3H]D3 with high affinity (Kd = 0.54 m), and the receptors exhibited a sedimen- tation constant of 3.7 S in a hypertonic sucrose gradient. The uptake of 1,25(OH)z[3H]D3 the centrifuged X for 30 an L5-40 Beckman ultracentrifuge. The supernatant was transferred to a scintillation vial and radioactivity was measured. Specific uptake was calculated by subtracting the radioactivity of the tube with excess unlabeled 1,25(OH)zD3 from that of the tube without unlabeled 1,25(OH)2DS.

We recently demonstrated that 25-hydroxyvitamin D3-24-hydroxylase, which can be further induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is localized exclusively in the proximal nephron of the vitamin Dreplete rat kidney. These data and the proposed mode of action of 1,25(OH)& predict the presence of a receptor for 1,25(OH)&3 in the proximal nephron. However, autoradiographic studies failed to detect significant nuclear uptake of 1,25(0H)~D3[~H]D~ in the proximal nephron.
To localize and characterize receptors for 1,25-(OH)2D3 along the nephron, preparations of both microdissected defined nephron segments and isolated tubules from the whole kidney of vitamin D-deficient rats were incubated with 1,25(OH)2[3H]D3, and specific uptake of the sterol was assessed in the cytosol preparation of tubular cells sonicated in a hypertonic solution. The preparation of isolated tubules incubated for 1 h a t 37 "C accumulated 1,25(OH)2[3H]D3 with high affinity (Kd = 0.54 m), and the receptors exhibited a sedimentation constant of 3.7 S in a hypertonic sucrose gradient. The uptake of 1,25(OH)z[3H]D3 was also examined in three defined single nephron segments: proximal convoluted tubules, medullary thick ascending limb of Henle's loop, and collecting tubules. Both proximal convoluted tubules and medullary thick ascending limb of Henle's loop showed a significant uptake of 1,25-(OH)2[3H]D3, 29.9 f 8.1 and 20.8 f 4.5 fmol/lOOO-mm tubule length, respectively, while collecting tubules showed no significant uptake. The receptors in both segments sedimented at 3.7 S in sucrose gradient. These data demonstrate the presence of 1,%(OH)2D3 receptors in both proximal and distal nephron.
* This work was supported by the Veterans Administration and in part by National Institutes of Health Grants AM-21351, AM-14750, and AM-20919. A portion of the present study was presented at the Fifth Workshop on Vitamin D, Williamsburg, VA, February 14- 19,1982, and appeared as an abstract in the abstract book of the Workshop. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ The mechanism of action of 1,25(OH)zD3 involves binding of this hormone to cytoplasmic receptors and migration of the steroid-receptor complex to the nucleus leading to the induction of hormone-specific protein synthesis (1)(2)(3)(4). Although the intestine and bone have generally been considered to be the major target organs, certain data suggest that 1,25(OH)2D3 may also act on the kidney (5,6). Recent studies demonstrated the presence of receptors for 1,25(OH)zD3 in the kidney of mouse (7), rat (a), and chick (9), as well as the nuclear transfer of the 1,25(OH)zD3-receptor complex in mouse kidney (10). It is also known that at least two distinct proteins, z.e. 25(OH)D3-24-hydroxylase and calcium-binding protein, are induced by 1,25(OH)z& in the kidney (1)(2)(3).
Recent autoradiographic data demonstrated that the nuclear uptake of 1,25(0H)~[~H]D~ in the kidney is predominantly localized to the distal nephron and to some extent to the glomerulus (11). These data are consistent with findings of the exclusive localization of vitamin D-dependent, calciumbinding protein in the distal nephron by immunohistochemical methods in the kidney of mouse (12), rat (13), and chick (14). Another protein induced by 1,25(OH)zD3, 25(OH)D3-24hydroxylase, has been exclusively localized to the proximal nephron ( 15).
Therefore, it seems reasonable that either a receptor for 1,25(OH)zD3 or another mechanism to recognize 1,25(OH)zD3 must be present in the proximal nephron in order to transfer the hormonal signal to the site of the protein synthesis. The present study was carried out aimed at the localization of receptors for 1,25(OH)zD3 along the nephron using defined single nephron segments; this was done to obtain further insight into the specific sites of action of 1,25(OH)zD3 in the kidney. Animals-Male weanling Holtzman rats were maintained for 3-5 weeks on a vitamin D-deficient diet containing 0.45% calcium and 0.3% phosphorus (15).

Materials-l,25-Dihydroxy-[26,27-methyl-3H]-vitamin
Preparation of Isolated Tubular Suspension-Isolated tubules were prepared according to a modification of previously reported methods (16). The animals were anesthetized with intraperitoneal sodium pentobarbital, 40 mg/kg of body weight. The superior mesenteric artery was ligated, and, immediately before perfusion of the kidneys, the aorta was ligated above the renal arteries. The kidneys were then perfused with 30 ml of chilled KRB buffer, pH 7.4, equilibrated with 95% oz/5% Con. The kidneys were removed, minced, and

1,25(OH)zD3 Receptors in Proximal and Distal Nephron 13429
incubated in the KRB buffer containing 0.1% collagenase (Sigma) at 37 "C for 45 min with gentle shaking in a 25-ml Erlenmeyer flask. At the end of incubation, the incubation mixture was gently pipetted and filtered by a nylon mesh. The filtrate was centrifuged at 750 X g for 30 min. The pellet was washed in Hanks' solution three times, and the final pellet was resuspended in Hanks' solution containing 8.3 mM glucose. This preparation represents a mixture of isolated fragments of tubules of the whole kidney. Preparation of Defined Single Nephron Segments-Perfused kidneys were removed and sliced (approximately 0.5-1.0 mm in thickness) according to methods that have been reported in detail elsewhere (15,17). Slices were incubated fo 30 min at 30 "C in KRB buffer containing 8.3 mM glucose, 0.1% bovine serum albumin, and 0.1% collagenase, with constant bubbling with 95% 02/5% COS. Kidney slices were then rinsed three times with 30-50 ml of ice-cold Hanks' solution, and defined single nephron segments were dissected freehand under a stereomicroscope in the ice-cold Hanks' solution.
Uptake solution containing defined single nephron segments was added to the tube. Incubation was carried out at 37 "C for 1 h with gentle shaking. After centrifugation at 3,000 X g for 5 min, the supernatant was discarded and the pellet was washed three times with 3.5 ml of isotonic solution containing 0.25 M sucrose, 25 mM KCI, 5 mM MgC12, 1 mM EDTA, 12 mM thioglycerol, 50 mM Tris-HCI, and 1 mg/ml of bovine serum albumin, pH 7.4. The pellet was resuspended in a hypertonic solution containing 0.3 M KCI, 0.5 mM dithiothreitol, 1.5 mM EDTA, and 10 mM Tris-HC1, pH 7.4 (18,19). The suspension was sonicated by a 15-s burst (Heat System, Ultrasonics, Inc.), and the sonicate was centrifuged at 105,000 X g for 30 min in an L5-40 Beckman ultracentrifuge. The supernatant was transferred to a scintillation vial and radioactivity was measured. Specific uptake was calculated by subtracting the radioactivity of the tube with excess unlabeled 1,25(OH)zD3 from that of the tube without Sucrose Density Gradient Analysis-The sonicate was centrifuged at 105,000 X g for 1 h. The supernatant (cystolic fraction) was layered onto 4-24% sucrose density gradients, prepared by layering 0.65 ml each of 24, 20, 16, 12, 8, and 4% sucrose in the hypertonic medium, and was centrifuged in an SW 50.1 rotor at 50,000 rpm for 24 h in an L5-75 Beckman ultracentrifuge. Fractions (30 X 0.16 ml) were collected from the top of the tube using Buchler Auto-densi Flow, and were counted for radioactivity by scintillation spectrometry. ["C] Ovalbumin (3.7 S) was used as a gradient marker.
Receptors in the Isolated Single Nephron Segments-The uptake of 1,25(OH)J3H]DS in the presence or absence of unlabeled 1,2510H)~Da and the sucrose density gradient analysis were performed using microdissected PCT, MTAL, and collecting tubules in the same way as described above. Each incubation contained approximately 500-1000 mm total length of the specific tubule segment, and the data were expressed as femtomoles of 1,25(OH)2[JH]D:I internalized per 1000 mm tubular length.
Other Assays-Protein concentration of the tubular preparations was determined by the Lowry method (20).  (Fig. 3).

Saturation Analysis-
Density Gradient Analysis-The sedimentation properties Da uptake by isobation, the tubular fragments were washed, sonicated, centrifuged, lated tubular preparations of the whole kidney. A, aliquots of and the radioactivity of the 105,000 X g supernatant was counted. the suspension of tubular fragments (2.5 mg of protein/0.5 ml of Nonspecific uptake was subtracted from total uptake to obtain specific medium) were incubated for 1 h at 37 "C with increasing concentra-uptake

1,25(OH)~Ds Receptors in Proximal and Distal Nephron
of the binding protein for 1,25(OH)2D3 in the 105,000 X g supernatant, examined by sucrose density gradient analysis, is shown in Fig. 4. The re~eptor-l,25(OH)2[~H]D3 complex was found to sediment at approximately 3.7 S in sucrose gradients prepared in the hypertonic buffer (Fig. 4A). However, when a tubular preparation was incubated with 25(OH)["H]DB, a radioactive peak appeared at 5.8 S, a value corresponding to the sedimentation constant for binding protein for 25(OH)D3. This peak was displaced in the presence of a 200-fold excess of unlabeled 25(OH)Da. These results again demonstrate that tubular cells can accumulate both 1,25(OH)2D3 and 25(OH)Da; however, the proteins to which these metabolites bind are different.

1,25(0H)@H]D3
Uptuke by Defined Single Nephron Segments-From the results described above, our assay system seems sensitive enough to localize 1,25(OH)2D3 uptake in the single nephron segments. Thus, we extended our methods attempting to quantify the amount of receptor in defined single nephron segments. We chose three nephron segments, i.e. PCT, MTAL, and collecting tubules. The MTAL is the segment where the nuclear uptake of 1,25(OH)zD3 has been detected by autoradiography. This segment is the easiest to dissect and collect in large quantity among the distal nephron segments, including cortical thick ascending limbs of Henle's loop and distal convoluted tubules. The PCT is the segment where 25(OH)D3-24-hydroxylase is localized; however, earlier autoradiographic data suggested little nuclear uptake of 1,25(OH)2D3. The collecting tubules is a segment where neither 1,25(OH)zD3 uptake nor 25(OH)D3-24-hydroxylase have been detected. Because of the technical difficulty of dissecting sufficient quantities of these segments from the same animal during a reasonable period of time, i.e. a few hours, we studied only two segments, either PCT and MTAL or MTAL and collecting tubules, in any given experiment using the same rat. Results are summarized in Table I. Th.e MTAL showed significant uptake of 1,25(OH)2["H]D3, 20.8 k 4.5 fmo1/1000 mm tubular length/h. The PCT also showed significant uptake of 1,25(OH)2[3H]D3, quantities comparable to those obtained in the MTAL. By contrast, collecting tubules showed little or no uptake. In experiments 4 and 6, this segment showed a significant uptake of 1,25(OH)z["H]D3, although this was much less than in the MTAL. In experiment 5, no significant uptake was
Nephron segments Experiment Mean f S.E.  To ascertain whether the uptake of 1,25(OH)2["H]D3 by the PCT or MTAL is mediated by a specific receptor, density gradient analysis was performed in the same manner described above for the tubular preparations from the whole kidney. As shown in Fig. 5, A and B, the radioactivity in both segments was found in fractions corresponding to a sedimentation constant of approximately 3.7 s. The radioactivity found in this peak was 70.6 and 78.9% of the total 1,25(OH)2[3H]D3 accumulated in the PCT and the MTAL, respectively. These results clearly demonstrate the presence of specific receptors in both PCT and MTAL.

DISCUSSION
The presence of receptors for 1,25(OH)zD3 has been well documented in the kidney of mouse (7), rat (8), and chick (9). Recent studies have demonstrated that vitamin D-dependent, calcium-binding protein is exclusively localized in the distal nephron in the kidney of chick, rat, and mouse (12)(13)(14). In addition, the nuclear uptake of 1,25(OH)2[3H]D3 injected in vivo in vitamin D-deficient rats is limited, by autoradiography, to the distal nephron; there was a small amount of uptake of 1,25(0H)~[~H]D3 in the glomerulus (11). These data strongly suggest that the distal nephron is a target site of 1,25(OH)2D3. On the other hand, we have demonstrated that 25(OH)D3-24hydroxylase is exclusively localized to the proximal tubules (15). Since induction of 25(OH)D3-24-hydroxylase by 1,25-(OH)*D3 in the kidney has been shown to require new protein synthesis (21, 22), it is reasonable to assume that there may be a receptor or similar mechanism to mediate the action of 1,25(OH)2D3 to induce synthesis of the new protein, 25(OH)D3-24-hydroxylase. To clarify this point, we attempted to localize receptors for 1,25(OH)zD3 along the rat nephron using the uptake of 1,25(OH)2['H]D3 by isolated tubules or defined single nephron segments.
The present results clearly demonstrate that receptors for 1,25(OH)zD3 are localized both in the distal nephron and in the proximal nephron. The presence of the receptor in the distal nephron is consistent with earlier data of the exclusive localization of calcium binding protein inducible by vitamin D (12)(13)(14) and the nuclear uptake of 1,25(OH)2[3H]D:3 in that part of nephron (11). The presence of the receptor in the proximal nephron is at variance from autoradiographic data (11). The reason why autoradiography failed to demonstrate the nuclear uptake of 1,25(OH)2[3H]D3 in the proximal nephron is not known. It should be noted that the absence of uptake of radioactivity in autoradiography does not necessarily rule out the presence of the receptor in the portion of the nephron. In fact, Stumpf et al. (11) indicated that they observed nuclear uptake of 1,25(OH)$H]D3 to some extent in the proximal tubular cells in some experiments.
It is possible that the time course of nuclear uptake of 1,25(OH)2D3 and/or the metabolism of the receptor-1,25-(OH)2D3 complex may be different in the proximal nephron from those of the distal nephron. The 105,000 X g supernatant of the sonicates in hypertonic solution contains not only cytosol fraction but also some part of the chromatin-bound receptor fraction when measured separately in the tubular preparation from whole kidney; approximately 30% of the internalized 1,25(0H)2[3H]D3 was transferred to the nuclear fraction during 1 h of incubation at 37 "C. However, the quantities of the PCT or MTAL collected in one experiment are very limited and are not sufficient to allow measurement of the separate binding of 1,25(OH)2D3 in nuclear and cytosolic fractions. Nonetheless, the present data provide direct evidence for the presence of 1,25(OH)~D3 receptors in both the proximal and distal nephron of the rat. The results strongly support the thesis that two distinct proteins can be induced by 1,25(OH)& 25(OH)D3-24-hydroxylase, and a calciumbinding protein, each of which is present in the proximal or distal nephron, respectively.