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Am J Physiol Regul Integr Comp Physiol 290: R560-R567, 2006. First published September 29, 2005; doi:10.1152/ajpregu.00279.2005
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Molecular Mechanisms Linking Salt to Hypertension

Modulation by dietary sodium intake of melanocortin 3 receptor mRNA and protein abundance in the rat kidney

Xi-Ping Ni, Aditi Bhargava, David Pearce, and Michael H. Humphreys

Division of Nephrology, San Francisco General Hospital and Department of Medicine, University of California San Francisco, San Francisco, California

Submitted 18 April 2005 ; accepted in final form 21 September 2005


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{gamma}-Melanocyte stimulating hormone ({gamma}-MSH) is a circulating natriuretic peptide hormone derived from proopiomelanocortin (POMC); its concentration in plasma and pituitary POMC mRNA abundance, increase in rats ingesting a high-sodium diet (HSD, 8% NaCl) compared with a low-sodium diet (LSD, 0.07% NaCl). RT-PCR of rat kidney RNA demonstrated reaction products of the expected size in both cortex and medulla for MC3-R, MC4-R, and MC5-R mRNA; no signal for MC1-R or MC2-R was detected. Relative to beta-actin or cyclophilin, abundance of the three receptor transcripts after 1 wk of the LSD was approximately equal in both cortex and medulla. After 1 wk of the HSD, mRNA abundance of MC4-R and MC5-R was unchanged, whereas that of MC3-R in medulla more than doubled, the ratio of MC3-R/beta-actin signal increasing from 0.38 ± 0.04 on LSD to 0.84 ± 0.04 on HSD (P < 0.001). No significant increase occurred in the cortex. The increase in MC3-R expression induced by dietary sodium was observed in inner medullary collecting duct (IMCD) cells isolated from the kidneys of HSD rats, suggesting that these cells were the major site of receptor expression in the medulla. Immunoblots of whole medullary and IMCD cell homogenates detected MC3-R immunoreactive protein; its expression was twice as great in samples from HSD vs. LSD rat kidneys, paralleling the increase in MC3-R mRNA abundance on the HSD. No changes in MC4-R or MC5-R protein expression were observed. Incubation of IMCD cell suspensions with increasing concentrations of {gamma}2-MSH led to increased cAMP accumulation, with values from rats on the HSD being roughly double the values from LSD rats. Intrarenal infusion of {gamma}2-MSH (500 fmol/min) increased sodium and cAMP excretion from the infused but not contralateral kidney of HSD rats, while having no effect in LSD rats. These data show that MC3-R is expressed in rat IMCD cells in a manner modulated by dietary sodium intake. Because MC3-R is the receptor with which {gamma}-MSH interacts, our findings suggest the existence of a sodium-regulating system, activated in response to a HSD, which increases urinary sodium excretion to balance the high-sodium intake.

{gamma}-melanocyte stimulating hormone; sodium excretion; inner medullary collecting duct; peptide hormone; sodium balance; cyclic AMP

{gamma}-MELANOCYTE STIMULATING HORMONE ({gamma}-MSH) is a natriuretic peptide derived, like {alpha}- and beta-MSH, from processing of proopiomelancortin (POMC) in the pituitary intermediate lobe (IL) from which it is secreted into the circulation (1, 9, 29). Three forms of {gamma}-MSH have been identified: {gamma}1-MSH has 11 amino acids with a COOH-terminal amidation, {gamma}2-MSH is identical to {gamma}1-MSH but with a COOH-terminal glycine added, and {gamma}3-MSH has an additional COOH-terminal extension of 13 amino acids; these structural differences have been suggested to underlie differing actions of the {gamma}-MSH peptides (10, 31, 34). MSH peptides exert their functions by interacting with a family of five transmembrane melanocortin receptors, termed MC1-R to MC5-R, which are linked to adenylate cyclase and possibly other signaling mechanisms (9, 28, 31, 34). Of the five MC-Rs, {gamma}-MSH has the highest affinity for MC3-R, and it is likely the natural ligand for this receptor (27).

The {gamma}-MSH system appears to be an important regulator of salt balance and blood pressure in rodents (13). Exposure to a high-sodium diet (HSD) results in a more than doubling of plasma {gamma}-MSH concentration, as well as an increase in IL {gamma}-MSH content and the mRNA of POMC compared with rats on a low-sodium diet (LSD) (3, 20, 21). The HSD also increases the IL mRNA abundance of two proconvertase enzymes involved in the processing of POMC into {gamma}-MSH (3). Induction of {gamma}-MSH deficiency, either pharmacologically or in a mouse genetic model, results in marked salt-sensitive hypertension, which is rapidly corrected with administration of the peptide (13, 21, 24). Mice with targeted disruption of the MC3-R (2) also develop salt-sensitive hypertension, which, however, is not ameliorated by {gamma}-MSH infusion (24).

Renal expression of MC3-R has been observed in human kidney (6), but its localization in cortex vs. medulla has not been examined, nor has its specific role in mediating the natriuresis caused by {gamma}-MSH. In the present study, we used RT-PCR and immunoblotting to demonstrate the expression of MC-Rs in rat kidney and evaluate whether the expression is modified by dietary sodium content. Our results indicate that mRNA and protein for MC3-R, MC4-R, and MC5-R exist in rat kidney and that only medullary MC3-R mRNA and protein abundance are increased in response to high dietary sodium intake. This increase in receptor expression appears functionally significant in that the natriuresis resulting from infusion of {gamma}-MSH is amplified in rats on the HSD. These results, along with other data showing dietary sodium-mediated increases in pituitary content and plasma concentration of {gamma}-MSH, suggest that parallel upregulation of both this natriuretic peptide and its renal receptor is part of the coordinated response to the stimulus of a high dietary sodium intake.


   METHODS
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 ABSTRACT
 METHODS
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We studied male Sprague-Dawley rats weighing 220 to 280 g; they were obtained from Charles River Laboratories (Hollister, CA). The protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.

Animal groups and tissue preparation. Rats were divided into three groups. One group was fed a normal-sodium diet (0.40% NaCl, NSD; Purina Mills Purified Diet 5001), another an LSD (0.07% NaCl, Purina Mills Purified Diet 5755, catalog no. 46843), and the third fed a HSD (8% NaCl, Purina Mills catalog no. 32892). Rats were killed by decapitation 1 wk after being on the different diets, and 3 ml of trunk blood were collected into a chilled vial containing 500 KIU aprotinin and 5 mg EDTA (Vacutainer tubes, Becton Dickinson, Franklin Lakes, NJ). The blood was centrifuged at 4°C, and the plasma was decanted and stored at –70°C until assayed for plasma {gamma}-MSH concentration. Kidneys and whole brain were excised from each animal. The kidney was then divided into cortex and medulla. These tissues were quickly diced into small pieces with a razor on ice and immediately put into liquid nitrogen and stored at –70°C. The adrenal gland was also excised from the NSD group and treated by the same protocol as described above to serve as a positive control for MC2-R.

Total RNA isolation and RT-PCR. The RNA STAT-60 method (Tel-Test, Inc, Friendswood, TX) was employed for RNA isolation from the frozen tissues and from a mouse melanoma cell line (Cloudman S91 melanoma, CCL-53.1, American Type Culture Collection, Manassas, VA). The tissue was homogenized with a polytron (Brinkmann Instruments, Westbury, NY) in 1 ml RNA STAT-60 per 50–100 mg tissue. RNA extraction was done by mixing 1 vol of the homogenate with 0.2 vol. of chloroform; then, 0.5 vol of isopropanol was used to precipitate the RNA. The pellet was washed with 75% ethanol and stored at –70°C. Sequences of the specific oligonucleotide primers were designed using the published sequences for MC1-R to MC5-R (Table 1).


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  		Table 1. Primers used for PCR amplification of melanocortin receptors and controls

 
One microgram of total RNA from each tissue sample was reverse transcribed at 42°C for 1 h into cDNA first strands, and PCR was performed with the RNA PCR core kit (Applied Biosystems, Foster City, CA) in a model 2700 thermal cycler (Applied Biosystems). The amplification conditions were determined empirically, and the products were analyzed on 1.2% agarose gel, photographed, and sequenced to confirm their identity (Biomolecular Resource Center, University of California, San Francisco, CA). The signal intensity was quantitated by means of pixel densitometry and normalized to the cyclophilin or beta-actin signal.

These procedures were repeated using freshly isolated and dispersed inner medullary collecting duct (IMCD) cells from rats on the LSD or HSD. Cells were obtained by collagenase digestion using the method of Zeidel et al. (35), as described for our laboratory (25, 30).

Immunoblotting. Slices of kidney cortex and medulla and freshly isolated IMCD cells were added to cold lysis buffer, 3 ml/g tissue (RIPA buffer, Santa Cruz Biotechnologies, Santa Cruz, CA) containing 1 x PBS, 1% igepal CA-63, 0.5% sodium deoxycholate, and 0.1% SDS (Sigma, St. Louis, MO), to which were added 100 µg/ml PMSF and 45 µg/ml aprotinin. The suspension was homogenized using a polytron at 0°C and centrifuged at 10,000 g at 4°C for 10 min. The supernatant was recentrifuged, and the second supernatant was measured for protein content using the dye-binding method of Bradford (Bio-Rad Laboratories, Hercules, CA) and stored at –20°C. Aliquots (40 µg protein) were size-fractionated on precast 4–20% gradient SDS-polyacrylamide gels in an Amersham SE 280 vertical gel electrophoresis unit at 40 mA for 1 h and electrotransferred to nitrocellulose membranes. These membranes were then blocked with 5% dried milk in PBS, pH 7.5, containing 0.1% Tween 20 for 1 h at room temperature, rinsed, and incubated for 1 h with the primary antiserum, raised against rat MC-Rs (MC3-R, Santa Cruz Biotechnology; MC4-R and MC5-R, Chemicon International, Temecula, CA) at a dilution of 1:800 for MC3-R; 1:300 for MC4-R; and 1:400 for MC5-R. Bound antibody was visualized using horseradish peroxide-conjugated donkey anti-rabbit secondary antibody diluted 1:1,000 for 30 min (RPN 2108 ECL kit; Amersham Arlington Heights, IL); the chemiluminescent signal was detected on X-ray film and quantitated by densitometric scanning.

Radioimmunoassay. For {gamma}-MSH assay, the frozen plasma samples were thawed on ice and extracted and eluted through Sep-Column chromatography cartridges (Peninsula Laboratories, Belmont, CA) as we have reported previously (17, 20). The eluate was lyophilized and stored at –70°C until assayed, as described previously (17, 20, 23) using a commercially available RIA kit (Peninsula Laboratories, Belmont, CA) with 125I-labeled {gamma}2-MSH as a tracer. Characteristics of this assay have been described (3, 21, 23). Results are expressed as femtomoles per milliliter of plasma.

{gamma}-MSH-dependent cAMP accumulation by isolated IMCD cells. Aliquots of freshly dispersed IMCD cells obtained from LSD and HSD rat kidneys as described above were suspended in 350 µl of buffer containing (mmol/l) of the following: 124 NaCl, 5 KCl, 1 CaCl2, 0.4 MgSO4, 1 Na2HPO4, 50 HEPES, and 7.5 glucose. In addition, we added 1 mmol/l 1,10-phenanthroline and 0.5 µg/ml leupeptin as protease inhibitors and 1 mmol/l 3-isobutyl-1-methylxanthine as a phosphodiesterase inhibitor. Cells were preincubated for 10 min at 37°C in a shaking water bath. Incubation was started by adding {gamma}2-MSH (Peninsula Laboratories, Belmont, CA) at a concentration of 10–9 to 10–6 mol/l or forskolin 10–4 mol/l and terminated after 15 min by adding 750 µl of ice-cold TCA. We used {gamma}2-MSH for these in vitro studies and the in vivo infusion studies described next because of our earlier work, indicating the predominance of this form of {gamma}-MSH in rat plasma (10, 14) and its demonstrated natriuretic effect (5, 17, 19). The precipitated protein was sedimented by centrifugation, and the pellet was dissolved in 1 N NaOH and assayed for protein concentration as described above. The supernatant fluid was extracted four times with five volumes of water-saturated ethyl ether to remove the TCA before being evaporated to dryness under a stream of air. Samples were stored at –70°C until assayed for cAMP content with the RIA kit TRK432 (Amersham, Piscataway, NJ). Results of duplicate determinations were averaged and expressed as picomoles accumulated per 15-min incubation per mg protein.

Intrarenal infusion of {gamma}-MSH. Rats fed the LSD or HSD were anesthetized with Inactin (100 mg/kg ip; Sigma, St. Louis, MO), and fitted with catheters in the jugular vein, carotid artery, bladder, and left ureter. The left renal pedicle was exposed via a flank incision and a curved 30-gauge needle attached to PE-10 tubing was inserted into the left renal artery with the tip directed toward the kidney. The rats received an intravenous infusion of normal saline at 1.8 ml/h throughout this surgical preparation and for the duration of the experiment. After 30–60 min of equilibration, urine was collected from the left ureteral catheter and the bladder, reflecting urine from the right kidney, for three 10-min intervals. {gamma}2-MSH was dissolved in normal saline and infused at a rate of 500 fmol/min in a volume of 7.5 µl/min, using a motor-driven syringe pump (Harvard Apparatus, Hollister, MA). Urine samples were collected again for three successive 10-min periods. Urine flow rate was determined gravimetrically, and urine sodium concentration was measured by flame photometry (model 943; Instrumentation Laboratories, Lexington, MA); sodium excretion (UNaV) was calculated as the product of the two. Urine was also assayed for cAMP and cGMP concentrations using radioimmunoassay kits (Amersham); the assays were conducted according to the manufacturer's instructions, and excretion rates were calculated from the product of concentration and urine flow.

Analysis of data. Results are expressed as means ± SE. Student's t-test for paired or unpaired data was used to assess differences between groups, and one-way and repeated-measures ANOVAs with the Bonferroni post hoc test were used for multiple comparisons among and within groups. A P value of <0.05 was taken to indicate a significant difference.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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Reverse transcription followed by PCR amplification using the primers depicted in Table 1 yielded PCR products of the predicted size for MC3-R, MC4-R, and MC5-R in both brain and kidney. In the kidney, they were detected in both cortex and medulla (Fig. 1). Their identity was confirmed by sequence analysis. No signal was detected in rat kidney for MC1-R or MC2-R, although an MC1-R signal was detected in mouse melanoma cells and an MC2-R signal was readily detected in rat adrenal (Fig. 2).


Figure 1
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  		Fig. 1. Photographs of gels showing PCR amplification products in rat brain and kidney tissue after reverse transcription of melanocortin receptors (MC)3-R, MC4-R, and MC5-R mRNA using the primers detailed in Table 1. A: brain and kidney cortex; B: brain and kidney medulla. Transcripts of the predicted sizes are observed for all three receptors in all of the tissues. Cyc, cyclophilin. Right column is size ladder in base pairs.

 

Figure 2
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  		Fig. 2. Gel photographs of RT-PCR results in Cloudman S9 melanoma cells (A) and rat adrenal gland (B) using primers for MC1-R and MC2-R as described in Table 1. A: primers for MC1-R; lane 1, molecular size marker in 100 base pair increments; 500 is indicated. Lane 2, rat renal cortex; lane 3, rat renal medulla; lane 4, melanoma cells. Signal for MC1-R product is clearly present in melanoma cells but not in kidney. B: lane 1, size ladder; lanes 2 and 4, renal cortex and medulla, respectively, MC1-R primers; lanes 3 and 5, renal cortex and medullar, respectively, MC2-R primers; lane 6, adrenal gland, MC1-R primers; and lane 7, adrenal gland, MC2-R primers. MC2-R reaction product is present in adrenal gland but not in renal cortex or medulla; MC1-R product is not present in any tissue.

 
To determine whether MC-R expression in rat kidney was modified by dietary sodium intake, we studied rats after 1 wk of ingesting the NSD, LSD, or HSD, using cyclophilin or beta-actin as an internal control. The results are shown in Fig. 3. Dietary sodium content did not alter mRNA expression for the receptors in renal cortical tissue. However, in rats ingesting a HSD for 1 wk, abundance of the reaction product in medullary samples was increased for MC3-R, while the product for beta-actin was unchanged. Scanning densitometry showed the ratio of MC3-R/beta-actin to be 0.38 ± 0.04 on the LSD, 0.39 ± 0.07 on the NSD, and 0.84 ± 0.04 units on the HSD (P < 0.001). Ratios of MC4-R/beta-actin and MC5-R/beta-actin were not significantly altered by dietary sodium intake (Fig. 3).


Figure 3
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  		Fig. 3. Quantitation by scanning densitometry of mRNA abundance of MC3-R, MC4-R, and MC5-R in rat renal cortex and medulla during ingestion of the low-, normal- (Nl), and high-sodium diets. Results have been normalized to beta-actin mRNA expression. The high-sodium diet increased the abundance of MC3-R mRNA but not that of the other two receptors. Values are presented as means ± SE of determinations from 5 rats. P < 0.001 vs. low and Nl, one-way ANOVA.

 
These data were obtained using RNA harvested from bulk medullary tissue. To define more precisely in what medullary cell type the dietary sodium-regulated expression of MC3-R was taking place, we repeated these studies using RNA isolated from freshly dispersed IMCD cells from kidneys of rats ingesting the HSD vs. LSD. The same effect of dietary sodium intake on MC3-R mRNA expression by these cells was observed as in the studies using RNA from whole medulla (Fig. 4). The ratio of MC3-R/beta-actin mRNA abundance was 0.47 ± 0.06 in cells from rats on the LSD, 0.39 ± 0.12 from NSD rats, and 1.14 ± 0.07 from HSD rats; the last value was significantly greater than either the LSD (P < 0.01) or NSD (P < 0.001) value (n = 4). These results indicate that MC3-R expression is strongly induced in IMCD cells, which are a major, and possibly the only, site of medullary MC3-R expression.


Figure 4
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  		Fig. 4. Photograph of gel showing mRNA abundance of MC3-R in IMCD cells harvested from kidneys of rats ingesting the low-sodium (LSD), normal-sodium (NSD), or high-sodium (HSD) diet for 1 wk. RNA was reverse-transcribed and amplified by polymerase chain reaction as described in METHODS. Also shown is the signal for beta-actin. Signal intensity for MC3-R is much greater in cells from the HSD rat.

 
To determine whether the increase in MC3-R mRNA abundance in response to the HSD was reflected in an increase in MC3-R protein, we carried out Western blot analysis of homogenates from both cortex and medulla of rat kidneys after 1 wk of HSD. Immunoreactive bands of the expected sizes for MC3-R (36 kd), MC4-R (40 kd), and MC5-R (41 kd) were demonstrated in both cortex and medulla. Quantification by densitometric scanning indicated that the protein abundance of MC3-R was increased significantly in medulla but not the cortex of rats ingesting the HSD; scanning density was 59 ± 12 units on the LSD and 75 ± 13 on the NSD but increased to 125 ± 10 on the HSD (P < 0.01, HSD vs. LSD; P < 0.05, HSD vs. NSD; P = NS, LSD vs. NSD, one-way ANOVA). These results roughly paralleled the increase in mRNA of this receptor observed on the HSD. Protein abundance of the other receptors was not affected by dietary sodium content in either the cortex or medulla (data not shown). We repeated these studies using lysates of IMCD cells taken from rats on the different diets; the results are shown in Fig. 5. These results matched those seen in whole medulla in that a significant increase in MC3-R protein abundance was observed in cells from rats on the HSD. However, no difference in protein expression was observed between LSD and NSD rats.


Figure 5
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  		Fig. 5. Protein abundance of melanocortin receptors in renal cortical and inner medullary collecting duct (IMCD) cell cytosol from kidneys of rats ingesting the low-, normal- (Nl), or high-sodium diet for 1 wk. Top: representative immunoblots of MC3-R (left), MC4-R (center) and MC5-R (right) in IMCD cell cytosol from kidneys of rats on the low-, normal-, or high-sodium diets. Bottom: results of scanning quantification of immunoblots of the three receptors on each of the different diets. MC3-R protein is increased in cytosol from IMCD cells but not the cortex of rats on the high-sodium diet. Dietary sodium content did not alter protein abundance of the other two-receptor protein. Values are presented as means ± SE of determinations from six rats. *Significantly greater than low or Nl values, P < .001 by one-way ANOVA.

 
We tested whether this increase in MC3-R expression by the HSD resulted in enhanced signaling through the receptor by measuring {gamma}-MSH-dependent cAMP accumulation in freshly dispersed IMCD cells isolated from rats on the HSD vs. LSD. The results are shown in Fig. 6. Increasing concentrations of {gamma}2-MSH in the medium led to a dose-dependent increase in cAMP accumulation by cells from both HSD and LSD rats. However, at any concentration of peptide, cAMP accumulation by HSD cells was roughly double the value observed in cells from LSD rats. Basal cAMP accumulation did not, however, differ between the two treatments. Incubation of cells with forskolin in the absence of {gamma}-MSH led to equivalent levels of cAMP accumulation by cells from both HSD- and LSD-treated rats, indicating that the dietary treatment did not alter the intrinsic capacity of these cells to produce cAMP by stimulation of adenylate cyclase.


Figure 6
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  		Fig. 6. {gamma}-MSH-dependent cAMP accumulation by IMCD cells isolated from kidneys of rats ingesting the HSD vs. the LSD diet for 1 wk. Results are presented as means ± SE of five separate determinations. *Significantly greater than the control value; P < 0.05 or greater; +HSD value significantly greater than LSD value, P < 0.05 or greater.

 
Plasma {gamma}-MSH concentration was measured in six rats ingesting the HSD and was more than double the value observed in six rats on the LSD (83.4 ± 18.7 vs. 31.0 ± 4.4 fmol/ml, P < 0.05). This result is similar to those reported earlier by us (3, 20, 21).

To test further the physiological relevance of these results, we infused {gamma}2-MSH into the left renal artery of anesthetized rats at 500 fmol/min, a rate calculated to achieve a concentration of the peptide in renal artery plasma of ~100 fmol/ml above the prevailing systemic plasma concentration. Intrarenal infusion of {gamma}2-MSH after 1 wk of the HSD led to an increase in ipsilateral sodium excretion (Fig. 7A) as we reported earlier (5, 17). No change in UNaV from the contralateral kidney occurred. In contrast to these results, {gamma}2-MSH infused into the left renal artery of rats on the LSD caused no increase in UNaV. Urinary cAMP excretion during {gamma}2-MSH infusion in HSD rats paralleled the increase in UNaV, although basal excretion was the same in both HSD and LSD rat kidneys (Fig. 7B). UcGMPV from the infused kidney of HSD rats did not change as a result of the infusion, being 14.3 ± 2.5 and 15.1 ± 4.4 pmol/min before, and 13.8 ± 1.8 and 16.2 ± 1.5 pmol/min during the infusion (P = NS).


Figure 7
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  		Fig. 7. Urinary sodium excretion (UNaV, µmol/min) (A) urinary cyclic AMP excretion (UcAMPV, pmol/min) (B) from left and right kidneys before and during infusion of {gamma}2-MSH (500 fmol/min) into the left renal artery of rats ingesting the HSD or LSD for 1 wk. The infusion commenced at 30 min as indicated by the arrow, and led to increases in both UNaV and UcAMPV from the infused kidney of HSD rats without affecting excretion from the contralateral kidney. The infusion did not increase excretion rates from either kidney of rats ingesting the LSD. Values are presented as means ± SE of measurements in 6 rats in each group. *Value significantly greater than control values, P < 0.01 or greater.

 

   DISCUSSION
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 ABSTRACT
 METHODS
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 DISCUSSION
GRANTS
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The {gamma}-MSH hormonal system appears to be an important mechanism involved in the maintenance of sodium homeostasis in rodents (13). The development of peptide deficiency, whether by pharmacologic inhibition of {gamma}-MSH secretion from the pituitary in rats (21) or by genetic impairment of POMC processing in mice (24), results in marked hypertension when ingesting a HSD; the hypertension is rapidly corrected by administration of exogenous {gamma}2-MSH. Hormone infusion increases UNaV in wild-type mice but not in mice with targeted deletion of Mc3r (24), the gene encoding the mouse receptor with which {gamma}-MSH interacts (27). This observation indicates that MC3-R is the receptor that mediates the natriuresis caused by the peptide and a priori suggests that it is expressed in kidney. Chhajlani (6) provided the first direct evidence for receptor expression in human kidney, although not indicating its localization in glomerular, tubular, or vascular structures or identifying its function. Our studies offer direct evidence of both cortical and medullary expression of MC3-R mRNA and protein in rat kidney and further localize this medullary expression to epithelial cells of the renal inner medulla. Importantly, expression of medullary mRNA and protein is upregulated in rats on the HSD, compatible with its role in mediating the natriuretic effect of {gamma}-MSH. We also found evidence for MC4-R and MC5-R mRNA and protein expression in rat kidney, although these were not altered by dietary sodium intake. On the other hand, we could not detect message expression of MC1-R or MC2-R.

MC1-R is the melanocortin receptor mediating the effect of {alpha}-MSH on skin pigmentation. This peptide is also natriuretic (12, 26), and Kohda et al. (16) reported immunodetection of the receptor protein in rat kidney. Our inability to detect renal expression of this receptor does not reflect technical problems related to primer selection or improper experimental conditions, as we were able to observe the signal as expected in RNA from a melanoma cell line. It is possible that the previous result (16) reflects interaction of the antibody used for immunolocalization with another MC-R protein. Neither Chhajlani (6) nor Mountjoy et al. (22) detected renal expression of MC1-R mRNA. It also seems unlikely that MC2-R, which mediates the effects of ACTH on adrenal steroidogenesis, is expressed in rat kidney. Our primers and experimental conditions were successful in detecting a signal in rat adrenal gland but could detect no such signal in kidney.

We carried out these experiments initially using cyclophilin as a control for expression of a "housekeeping" gene. We became aware of a report that sodium intake affected the renal expression of a cyclophilin-like protein (15). Although we could document no change in expression of cyclophilin mRNA on the different diets (data not shown), we repeated our studies using beta-actin as a control. There are data indicating that dietary sodium intake does not affect beta-actin expression in a number of tissues, including the kidney (11, 32, 33). There were no major differences in the results normalizing mRNA abundance of the melanocortin receptors to either marker.

The observation that sodium loading increases not only the synthesis and secretion of {gamma}-MSH from the pituitary (3, 20, 21), but also the renal expression of its receptor MC3-R mRNA and protein suggests that this system is an important contributor to the maintenance of sodium balance under conditions of dietary sodium surfeit. In many endocrine systems, increased expression of a hormone leads to receptor downregulation, and the parallel upregulation of both ligand and receptor in our study attests to the potential importance of this, until recently, unrecognized natriuretic hormone system in sodium homeostasis. This was indicated functionally by the in vivo studies in which intrarenal infusion of {gamma}2-MSH led to a robust natriuresis in rats fed the HSD, with increased renal expression of MC3-R mRNA and protein, whereas in rats on the LSD, with lower expression of the receptor, the same infusion of exogenous peptide did not increase UNaV from the very low basal rate observed in these rats. It must, of course, be recognized that the LSD in all likelihood activated potent antinatriuretic pathways such as the renin-angiotensin-aldosterone system and renal sympathetic nerve activity, which could have obscured a natriuretic response to peptide infusion. Indeed, the response to other natriuretic stimuli such as infusion of atrial natriuretic peptide, volume expansion, or acute unilateral nephrectomy, is conditioned by the prior sodium intake and plasma volume of the experimental animal (4, 7, 8, 18). However, the development of marked hypertension on the HSD in rats and mice with {gamma}-MSH deficiency (21, 24) or in mice with disruption of Mc3r (24) is testimony to the importance of this system in sodium metabolism under conditions of sodium excess.

Our data suggest that these dietary sodium-induced changes in MC3-R expression in IMCD cells have functional significance. In most systems so far examined, the melanocortin receptor family signals through stimulation of adenylate cyclase with an increase in intracellular cAMP (9, 28, 34). We observed that incubation with {gamma}-MSH led to a dose-dependent increase in cAMP accumulation by freshly dispersed IMCD cells from kidneys of both HSD and LSD rats, but the accumulation was greater in cells from HSD rat kidneys. The concentrations of {gamma}-MSH used in these experiments were considerably higher than the concentration range found in the rodent circulation (~5 x 10–11 M), raising the concern that this reflects a pharmacological rather than a physiological stimulation of cAMP accumulation. Although we cannot dismiss this possibility with certainty, we do not think it to be the case. {gamma}-MSH is a notoriously labile peptide (19), and even though we added the peptidase inhibitors leupeptin and 1,10-phenanthroline to our incubation mix, it is possible that low concentrations of the peptide were inactivated before stimulating adenylate cyclase through activation of the MC3-R. In any case, the peptide clearly increased cAMP production by cells from HSD rat kidneys at each concentration tested to a greater extent than by cells from LSD animals. This seems specific to the peptide because basal rates of cAMP accumulation did not differ in the two preparations. Incubation of cells from HSD and LSD animals with the adenylate cyclase stimulator forskolin in the absence of {gamma}-MSH led to equivalent rates of cAMP accumulation, indicating that the different diets did not alter the intrinsic activity of this enzyme. We did not carry out a full dose-response curve to demonstrate saturation of {gamma}-MSH-dependent cAMP accumulation. However, to the extent that the results with forskolin reflect maximal cAMP generation, the value observed with 10–6 M {gamma}-MSH is nearly equal, suggesting that this concentration is close to saturation.

These results from the in vitro experiments were replicated and extended by our observations on the consequence of intrarenal peptide infusion in vivo. In these experiments, an infusion of {gamma}2-MSH calculated to achieve a physiologically relevant concentration of the peptide in renal plasma stimulated UcAMPV in rats preconditioned with the HSD but not in rats on the LSD, paralleling the consequences of the infusion on UNaV. Basal UcAMPV did not differ between the two groups, suggesting that other determinants of UcAMPV, such as vasopressin and parathyroid hormone, were not affected by the HSD, and the infusion had no effect on UcGMPV, indicating that the increase in UcAMPV was not a nonspecific effect of increased urine flow on cyclic nucleotide excretion but rather was specifically related to the peptide infusion. Urinary cyclic nucleotide excretion clearly reflects the complex effects of hormone action throughout the nephron; the in vitro studies in IMCD cells show a much clearer relationship between MC3-R expression and {gamma}-MSH-dependent cAMP accumulation. The failure of the peptide infusion to stimulate UcAMPV or natriuresis in rats on the LSD differs from these in vitro results, Although the basis for this difference is not clear, it is consistent with effects of the LSD on peptide-receptor binding or signaling through MC3-R, which are transient and not manifest in cells after harvesting and transfer to cell culture conditions.

In summary, we have demonstrated the presence of MC3-R mRNA and protein in both cortex and medulla of the rat kidney and have shown that expression of the receptor is increased during ingestion of the HSD. This receptor mediates the natriuretic actions of {gamma}-MSH. The upregulation of receptor expression in response to the HSD has functional significance in that there is an accelerated accumulation of {gamma}-MSH-dependent cAMP by IMCD cells from kidneys of HSD rats, and an amplification of the natriuresis resulting from peptide infusion. We have also detected the renal expression of MC4-R and MC5-R mRNA and protein; however, their abundance is not influenced by the HSD. The {gamma}-MSH system appears to function as a natriuretic system activated in response to a HSD; as such, it must operate in parallel with other natriuretic pathways, such as the atrial natriuretic peptide system to defend against progressive volume expansion in the face of high dietary sodium intake.


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 ABSTRACT
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Dr. Pearce was supported by Grants R-O1-DK-51151 and R-O1-DK-56695, and Dr. Humphreys by grant R-O1-HL-68871, from the National Institutes of Health.

Present address of A. Bhargava: Department of Surgery, 521 Parnassus Ave., Rm C-317, University of California San Francisco, San Francisco, CA 94143–0660.


   ACKNOWLEDGMENTS
 
Portions of this work were presented at the American Society of Nephrology 35th Annual Meeting, Philadelphia, PA, November, 2002 and the 37th Annual Meeting, St. Louis, MO, October, 2004 and have appeared in abstract form (J Am Soc Nephrol 13: 81A, 2002 and J Am Soc Nephrol 15: 15A, 2004).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Michael H. Humphreys, Box 1341, Univ. of California San Francisco, San Francisco, CA 94143 (E-mail: mhumphreys{at}medsfgh.ucsf.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
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