Adrenomedullin Inhibits Osmotic Water Permeability in Rat Inner Medullary Collecting Ducts

Adrenomedullin (ADM) is a vasodilator that causes natriuresis and diuresis. However, the direct effect of ADM on osmotic water permeability in the rat inner medullary collecting duct (IMCD) has not been tested. We investigated whether ADM and its ADM receptor components (CRLR, RAMP2, and 3) are expressed in rat inner medulla (IM) and whether ADM regulates osmotic water permeability in isolated perfused rat IMCDs. The mRNAs of ADM, CRLR, and RAMP2 and 3 were detected in rat IM. Abundant protein of CRLR and RAMP3 were also seen but RAMP2 protein level was extremely low. Adding ADM (100 nM) to the bath significantly decreased osmotic water permeability. ADM significantly decreased aquaporin-2 (AQP2) phosphorylation at Serine 256 (pS256) and increased it at Serine 261 (pS261). ADM significantly increased cAMP levels in IM. However, inhibition of cAMP by SQ22536 further decreased ADM-attenuated osmotic water permeability. Stimulation of cAMP by roflumilast increased ADM-attenuated osmotic water permeability. Previous studies show that ADM also stimulates phospholipase C (PLC) pathways including protein kinase C (PKC) and cGMP. We tested whether PLC pathways regulate ADM-attenuated osmotic water permeability. Blockade of either PLC by U73122 or PKC by rottlerin significantly augmented the ADM-attenuated osmotic water permeability and promoted pS256-AQP2 but did change pS261-AQP2. Inhibition of cGMP by L-NAME did not change AQP2 phosphorylation. In conclusion, ADM primarily binds to the CRLR-RAMP3 receptor to initiate signaling pathways in the IM. ADM reduced water reabsorption through a PLC-pathway involving PKC. ADM-attenuated water reabsorption may be related to decreased trafficking of AQP2 to the plasma membrane. cAMP is not involved in ADM-attenuated osmotic water permeability.


Introduction
Adrenomedullin (ADM) is a vasodilator peptide first extracted from human pheochromocytoma tissue in 1993 [1]. It is a 52-amino acid peptide that belongs to the calcitonin/calcitonin gene-related peptide (CGRP) family, which includes calcitonin, CGRP, and amylin. ADM receptors are type II G protein-coupled receptors [2], and each ADM receptor is formed by a complex of calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein 2 or 3 (RAMP2 or RAMP3). RAMPs conjugate with CRLR in the endoplasmic reticulum and facilitate the glycosylation and transport of CRLR to the cell surface [3,4]. ADM binds to the receptor of CRLR-RAMP 2 or 3, resulting in stimulation of adenylyl cyclase and phospholipase C (PLC) [5]. Subsequently, adenylyl cyclase activates cyclic adenosine monophosphate (cAMP) signaling pathways [1,2,6] and PLC activates cyclic guanosine monophosphate

Tubule Perfusion
IMs were transferred to a dissection dish and the terminal IMCDs microdissected in a dissection buffer at 17 • C. The dissecting solution contained (in mM): 125 NaCl, 25 NaHCO 3 , 2 CaCl 2 , 2.5 K 2 HPO 4 , 1.2 MgSO 4 , 5.5 glucose. The dissecting solution had a pH of 7.4 and an osmolality of 430 mosmol/kg H 2 O [22]. The perfusion and bath solutions were identical to the dissection medium in chemical composition, except that 5 mM raffinose was added to both the perfusate and the bath and an additional 70 mM NaCl was added to the bath when measuring osmotic water permeability to create a bath-to-lumen osmolality gradient of ∼140 mosmol/kg H 2 O. Both the perfusion and bath solutions had a pH of 7.4. The perfusion solution had an osmolality of 290 mosmol/kg H 2 O. The bath solution had an osmolality of 430 mosmol/kg H 2 O. All solutions were gassed continuously with 95% air and 5% CO 2 before and during the dissection and perfusion.
Single IMCDs were dissected, mounted on glass pipettes, and perfused as described [16]. In general, 45 min after warming the tubules to 37 • C in 2 mL of bath solution, two initial collections of 2-3 min at 6-8 nL/min were made. Treatments were added to the bath and tubules allowed to equilibrate for 20 min, then two further 2-3 min collections were made. To measure Pf, collected solutions were assayed for raffinose content by ultramicrofluorometry [23]; raffinose was used as a volume marker. Pf was calculated as described [16]. Biological variation in baseline osmotic water permeability between different animals has been recognized for many years [24]. Therefore, we use each tubule as its own control.
To assess the contribution of ADM to osmotic water permeability, Pf was measured in the absence of ADM, then ADM (100 nM) was added to the bath for 30 min after which 2 collections were made for raffinose determination.
To determine whether cAMP, PLC, or PKC are involved in regulating ADM-attenuated osmotic water permeability, SQ22536 (300 µM), U73122 (5 µM) and rottlerin (50 µM) were added to the bath for 20 min to inhibit cAMP, PLC, and PKC, respectively, and roflumilast (30 nM) was added to the bath for 20 min to stimulate cAMP after ADM-attenuated osmotic water permeability was measured. Then, 2 further collections were made for raffinose determination.

Tissue Incubation
To measure AQP2 phosphorylation, one of the two IMs from the same animal was assigned as a control; the other IM was assigned as a treatment. IMs were sectioned into ∼1 mm 3 tissue pieces and incubated in isotonic Hanks' balanced salt solution (HBSS). The dissecting HBSS solution contained (in mg/L): 400 KCl, 60 KH 2 PO 4 , 350 NaHCO 3 , 8000 NaCl, 90 Na 2 HPO 4 ·7H 2 O, 1000 dextrose anhydrous. The IM pieces were first incubated at 37 • C for 10 min in HBSS to establish base-line resting conditions, and then stimulated by 100 nM ADM in the treatment group. To test the effect of the inhibitors of cAMP, PLC, PKC, and cGMP, both the control and treatment groups were stimulated for 30 min with 100 nM ADM. Subsequently, SQ22536 (300 µM), U73122 (5 µM), rottlerin (50 µM), or L-NAME (5 µM) was added to the treatment group and incubation of all samples continued for 20 min. The reactions were terminated by placing the samples on ice and replacing the incubation solutions with ice-cold homogenization buffer (10 mM triethanolamine, 250 mM sucrose, 10% sodium dodecyl sulfate). Tissues were homogenized and analyzed for AQP2 phosphorylation by Western blot. AQP2 phosphorylation level was calculated as the ratio of phospho-protein/total protein to determine if the phosphorylation level per protein available changes.

RNA-Seq Analysis
Male Sprague-Dawley rats weighing 150-175 g, purchased from Charles River Laboratories, were used. Rats (n = 3) were sacrificed by decapitation. The IMs were dissected from kidney and used for RNA preparation. The total RNAs were extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) and purified using an RNeasy Mini Kit (Qiagen, Germantown, MD, USA). RNA sample quantity and quality were determined using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 bioanalyzer. Equal amounts of purified mRNA were transcribed to cDNA using a SMARTer PCR cDNA Synthesis Kit (Clontech, Waltham, MA, USA). Libraries were prepared according to Illumina's instructions; cDNA was fragmented by DNase I and ligated to Illumina adapters. These adapter-ligated cDNA fragments were amplified and sequenced on the Illumina HiSeq2000 sequencer. RNA-Seq data were analyzed by an Emory core using bioinformatic software (Tophat (v 1.3.3) and cuffdiff (v 1.3.0)). The mRNA expression signals were evaluated by RPKM (reads per kilobase million). AQP2 was used as IMCD marker, AQP1 as thin descending limb (tDL) marker.

Measurement of cAMP Level in Inner Medulla
To measure cAMP levels, one of the two IMs from the same animal was assigned as a control; the other IM was assigned as a treatment. The IM tissues were incubated with 100 nM ADM for 30 min. The rats were sacrificed by decapitation. The IMs were removed and quickly frozen with liquid nitrogen. Then the tissues were homogenized with TCA on ice (0-4 • C) and centrifuged at 1500× g for 10 min. The precipitates were removed, and TCA was extracted using water-saturated ether. The supernatants were collected and assayed for cAMP level. The cAMP level of the IM tissues was determined by ELISA using an enzyme immunoassay kit from Cayman Chemical Company (Ann Arbor, MI, USA).

Statistics
All data are presented as means ± SE. Data from tubule perfusion studies were analyzed using a paired Student's t-test [25]. Each perfused tubule serves as its own control, allowing a paired analysis of the data. Data from protein analysis studies were also analyzed using a paired Student's t-test. The criterion for statistical significance is p < 0.05.

ADM and Its Receptor Components (CRLR, RAMP2 and RAMP3) Are Expressed in the Inner Medulla
To determine if ADM and its receptor components are present in the IM, RNA sequencing analysis was performed on IM tissues. AQP2 was used as IMCD marker, AQP1 as tDL marker. Our results indicate that mRNAs for CRLR are detected. RAMP2 and RAMP3, but not RAMP1, are abundantly expressed in the IM. A low amount of ADM was detected in the IM (Table 1). IM tissue lysates were also subject to Western analysis and probed with antibodies to CRLR, RAMP2 and RAMP3. Our results show that the 53-kDa band of CRLR protein, the 50-kDa band of RAMP3 protein, and the 20-kDa band of RAMP2 protein are detected in the IM. CRLR and RAMP3 are expressed at a significant protein level. However, RAMP2 was expressed at a very low level ( Figure 1). The antibodies for CRLR, RAMP2, and RAMP3 were characterized in mice so mouse samples were used as positive controls (data not shown) to verify the Western blot results in rat tissues. Cells 2020, 9, x FOR PEER REVIEW 5 of 15 IM tissue lysates were also subject to Western analysis and probed with antibodies to CRLR, RAMP2 and RAMP3. Our results show that the 53-kDa band of CRLR protein, the 50-kDa band of RAMP3 protein, and the 20-kDa band of RAMP2 protein are detected in the IM. CRLR and RAMP3 are expressed at a significant protein level. However, RAMP2 was expressed at a very low level ( Figure 1). The antibodies for CRLR, RAMP2, and RAMP3 were characterized in mice so mouse samples were used as positive controls (data not shown) to verify the Western blot results in rat tissues.

ADM Decreases Osmotic Water Permeability
To determine whether ADM regulates osmotic water permeability, rat terminal IMCDs were first perfused under a basal condition without ADM and then perfused with ADM. ADM decreased Pf from 57 ± 6 to 42 ± 5 µm/s (n = 4, p < 0.05; Figure 2). To ensure that we were not observing a rundown phenomenon, we tested for time-related decreases in Pf and saw that Pf remained stable for up to 3 h (data not shown). In addition, we previously proved that 45-min incubation of tubules in the bath is enough to achieve a steady level of the basal water permeability [16].

ADM Decreases Osmotic Water Permeability
To determine whether ADM regulates osmotic water permeability, rat terminal IMCDs were first perfused under a basal condition without ADM and then perfused with ADM. ADM decreased Pf from 57 ± 6 to 42 ± 5 µm/s (n = 4, p < 0.05; Figure 2). To ensure that we were not observing a run-down phenomenon, we tested for time-related decreases in Pf and saw that Pf remained stable for up to 3 h (data not shown). In addition, we previously proved that 45-min incubation of tubules in the bath is enough to achieve a steady level of the basal water permeability [16]. IM tissue lysates were also subject to Western analysis and probed with antibodies to CRLR, RAMP2 and RAMP3. Our results show that the 53-kDa band of CRLR protein, the 50-kDa band of RAMP3 protein, and the 20-kDa band of RAMP2 protein are detected in the IM. CRLR and RAMP3 are expressed at a significant protein level. However, RAMP2 was expressed at a very low level ( Figure 1). The antibodies for CRLR, RAMP2, and RAMP3 were characterized in mice so mouse samples were used as positive controls (data not shown) to verify the Western blot results in rat tissues.

ADM Decreases Osmotic Water Permeability
To determine whether ADM regulates osmotic water permeability, rat terminal IMCDs were first perfused under a basal condition without ADM and then perfused with ADM. ADM decreased Pf from 57 ± 6 to 42 ± 5 µm/s (n = 4, p < 0.05; Figure 2). To ensure that we were not observing a rundown phenomenon, we tested for time-related decreases in Pf and saw that Pf remained stable for up to 3 h (data not shown). In addition, we previously proved that 45-min incubation of tubules in the bath is enough to achieve a steady level of the basal water permeability [16].

cAMP Regulates Osmotic Water Permeability Independently of ADM
To test ADM-attenuated osmotic water permeability through a cAMP pathway, IMCDs were first perfused with ADM, then perfused with SQ22536, an adenylyl cyclase inhibitor, in the presence of ADM. SQ22536 further decreased the ADM-attenuated Pf from 66 ± 10 to 58 ± 11 µm/s (n = 4, p < 0.05; Figure 4). To further identify the role of cAMP in ADM-attenuated osmotic water permeability, IMCDs were perfused with ADM, then perfused with roflumilast, a phosphodiesterase inhibitor, in the presence of ADM. Roflumilast increased the ADM-attenuated Pf from 73.42 ± 10.68 to 103.83 ± 5.48 µm/s (n = 3, p < 0.05; Figure 5).

cAMP Regulates Osmotic Water Permeability Independently of ADM
To test ADM-attenuated osmotic water permeability through a cAMP pathway, IMCDs were first perfused with ADM, then perfused with SQ22536, an adenylyl cyclase inhibitor, in the presence of ADM. SQ22536 further decreased the ADM-attenuated Pf from 66 ± 10 to 58 ± 11 µm/s (n = 4, p < 0.05; Figure 4). To further identify the role of cAMP in ADM-attenuated osmotic water permeability, IMCDs were perfused with ADM, then perfused with roflumilast, a phosphodiesterase inhibitor, in the presence of ADM. Roflumilast increased the ADM-attenuated Pf from 73.42 ± 10.68 to 103.83 ± 5.48 µm/s (n = 3, p < 0.05; Figure 5).

Inhibition of PLC Increases Osmotic Water Permeability
To test whether ADM-attenuated osmotic water permeability through a PLC pathway, IMCDs were first perfused with ADM, then perfused with U73122, a PLC inhibitor, in the presence of ADM.

Inhibition of PLC Increases Osmotic Water Permeability
To test whether ADM-attenuated osmotic water permeability through a PLC pathway, IMCDs were first perfused with ADM, then perfused with U73122, a PLC inhibitor, in the presence of ADM. U73122 increased Pf from 37 ± 8 to 54 ± 10 µm/s (n = 4, p < 0.05; Figure 7). Figure 6. ADM increases the cAMP level in rat inner medulla. Rat kidney inner medullas were treated with (1 kidney IM) or without (contralateral kidney IM) 100 nM adrenomedullin (ADM), then lysates prepared and analyzed by ELISA. Bars: mean ± s.e., n = kidneys from 7 individual rats, * = p < 0.05 control vs. ADM.

Inhibition of PLC Increases Osmotic Water Permeability
To test whether ADM-attenuated osmotic water permeability through a PLC pathway, IMCDs were first perfused with ADM, then perfused with U73122, a PLC inhibitor, in the presence of ADM. U73122 increased Pf from 37 ± 8 to 54 ± 10 µm/s (n = 4, p < 0.05; Figure 7).

Inhibition of PKC Increases Osmotic Water Permeability
To test whether ADM-attenuated osmotic water permeability through a PKC pathway, IMCDs were first perfused with ADM, then perfused with rottlerin, a PKC inhibitor, in the presence of ADM. Rottlerin increased Pf from 38 ± 7 to 52 ± 6 µm/s (n = 4, p < 0.05; Figure 9).

Inhibition of PKC Increases Osmotic Water Permeability
To test whether ADM-attenuated osmotic water permeability through a PKC pathway, IMCDs were first perfused with ADM, then perfused with rottlerin, a PKC inhibitor, in the presence of ADM. Rottlerin increased Pf from 38 ± 7 to 52 ± 6 µm/s (n = 4, p < 0.05; Figure 9).

Discussion
Water reabsorption is mainly regulated by vasopressin through cAMP-dependent signaling pathways [16,[26][27][28]. Recent studies demonstrate that local, paracrine, and autocrine hormones such as ADM are also involved in the regulation of fluid and electrolyte homeostasis [5,12]. ADM is expressed in the kidney [8,10]. However, limited studies have been done to elucidate the renal effect

Discussion
Water reabsorption is mainly regulated by vasopressin through cAMP-dependent signaling pathways [16,[26][27][28]. Recent studies demonstrate that local, paracrine, and autocrine hormones such as ADM are also involved in the regulation of fluid and electrolyte homeostasis [5,12]. ADM is expressed in the kidney [8,10]. However, limited studies have been done to elucidate the renal effect of ADM on water reabsorption. This study provides evidence that ADM decreases osmotic water permeability through PLC-PKC signaling pathways.
Previous studies show that ADM and ADM receptors are detected in the kidney [6,9]. But the expression of ADM receptors is not well defined. Analysis of mRNA showed that ADM mRNA is most abundant in the cortex. In contrast, mRNA for ADM receptors is most abundant in the IM [11]. But that study did not address which specific ADM receptor type, CRLR/RAMP2 or CRLR/RAMP3, is expressed in the kidney. Immunohistochemistry analysis showed that RAMP3 is mainly expressed in collecting duct cells, while RAMP2 is not expressed on tubular cells in normal kidney tissues [29]. However, the expression of CRLR was not tested in this immunohistochemistry analysis. To better understand the distribution of ADM receptors in rat IM, we analyzed the mRNAs for ADM, CRLR, RAMP2, and RAMP3. Our results show that mRNAs for ADM, CRLR, RAMP2, and RAMP3 are present in IM (Table 1). A low amount of ADM is detected, which suggests ADM exerts an autocrine and/or paracrine regulation mechanism in the IM. Our findings for the location of mRNA of ADM and its receptors are consistent with those reported by Knepper's group [30]. The small difference could come from different materials or dissected tubules vs. IM tissues. IM tissues contain IMCDs but also small amounts of interstitial tissues including capillary vessels. In addition, IM also contains a small portion of long limbs of Henle loop. We compared these two data sets: a low (marginal) amount of ADM was identified in both IMCD (Knepper's data) and IM (our data). We saw a low level of CRLR in IM but this was not seen by the Knepper group in IMCD. Neither we nor the Knepper group saw any RAMP1 in either IMCD or IM. We both observed RAMP2 (in IMCD and IM). RAMP3 is reported to be in a thin ascending limb by the Knepper group and we were able to see both RAMP3 mRNA and protein in IM.
We also performed Western blot using specific antibodies raised against CRLR, RAMP2 and RAMP3. Our results show a significant protein expression of CRLR and RAMP3. In contrast, RAMP2 is expressed at a very low level (Figure 1), which is consistent with the finding that RAMP2 protein is not expressed on tubular cells in the immunohistochemistry analysis [29]. Our results suggest that CRLR-RAMP3 is the predominant receptor that ADM interacts with rat IM. Although the mRNA for RAMP2 is present in rat IM, the translation of mRNA to protein may be blocked by unknown mechanisms. It is worthwhile to mention that the molecular weight of CRLR varies among various species depending on the level of glycosylation [31]. RAMP3 is also present in three different types: RAMP3 monomer (28 kDa), RAMP3 homodimer (50 kDa), and RAMP3 heterodimer (73-75 kDa) [32].
Our results indicate that the molecular weight of CRLR is 53 kDa and RAMP3 is a homodimer with a molecular weight of 50 kDa in rat IM.
Initially, ADM was identified as a cAMP stimulator via activation of adenylyl cyclase [1,2,6]. cAMP is involved in vasopressin-stimulated water reabsorption in the kidney [16,[26][27][28]. Given its ability to activate cAMP, we expected that ADM may also stimulate water reabsorption through cAMP. Therefore, we tested the osmotic water permeability in isolated perfused IMCDs. Surprisingly, ADM decreases osmotic water permeability in IMCDs ( Figure 2) despite an increase in cAMP level ( Figure 6), which supports the finding that intrarenal arterial infusion of ADM causes diuresis, not anti-diuresis [13,14]. ADM decreases the phosphorylation of AQP2 at serine 256, whereas it increases phosphorylation of AQP2 at serine 261 ( Figure 3). Phosphorylation of AQP2 at serine 256 promotes AQP2 trafficking to the apical plasma membrane [33], and phosphorylation of AQP2 at serine 261 is linked to ubiquitination [34]. This suggests that ADM decreases plasma membrane accumulation of AQP2 through phosphorylation at serine 256 and increases ubiquitination of AQP2 associated with phosphorylation at serine 261. The data in the present study demonstrated that ADM regulates osmotic water permeability independent of cAMP pathways even though ADM increases the cAMP level. To verify the role of cAMP in ADM-regulated water permeability, we tested whether an adenylyl cyclase inhibitor, SQ22536, affects ADM-attenuated water permeability. Our results show that inhibition of cAMP by SQ22536 further decreased ADM-attenuated water permeability (Figure 4). We also tested whether a phosphodiesterase inhibitor, roflumilast, affects ADM-attenuated water permeability. PDE4b specifically catalyzes cAMP hydrolysis. Roflumilast is a blocker of PDE4, which could lead to increased cAMP. Our results show that stimulation of cAMP by roflumilast increased ADM-attenuated water permeability ( Figure 5). Obviously, cAMP regulates water reabsorption in its own way and does not interfere with ADM-regulated water permeability. To confirm that the nonintervention of cAMP in ADM-attenuated water permeability is not due to the deficiency of cAMP, we tested cAMP production in the IM. Our result indicated that ADM significantly increases cAMP level ( Figure 6). However, we admit that we cannot tell IMCDs from thin limbs in the cAMP measurements in IM. Our result suggested that ADM increases the cAMP level in either IMCDs, or thin limbs, or both. Previous studies [6,11] showed that the cAMP level is increased in IMCDs in response to ADM. Therefore, it is likely that the ADM-stimulated cAMP level in this study results primarily, but not exclusively, from IMCDs.
The inconsistency of the role of cAMP in regulation of osmotic water permeability may be explained by the concept of compartmentalized cAMP signaling [35,36]. cAMP signaling pathways control a variety of cellular processes in a stimulation-specific manner [36]. ADM may stimulate a different cAMP pool from that stimulated by vasopressin. Thus, ADM-stimulated cAMP may not be able to activate PKA and further increase water reabsorption. The alternative explanation is that other signaling pathways stimulated by ADM have a robust inhibitory influence and counteract the stimulatory effect of cAMP on osmotic water permeability.
In addition to cAMP, ADM stimulates PLC through G protein-coupled receptors [5]. PLC further activates PKC and cGMP [5]. Previous studies indicated that ADM regulates some cellular functions in cAMP-independent pathways. ADM enhances cardiac contractility via cAMP-independent mechanisms including activation of PKC [37]. ADM induced vasorelaxation in the rat aorta and kidney via a NO-cGMP pathway [38]. To determine whether the decrease in osmotic water permeability by ADM is in response to the activation of PLC, the PLC inhibitor, U73122, was used to reverse the decreased osmotic water permeability. Data from isolated perfused tubules show that U73122 significantly increases ADM-attenuated water permeability (Figure 7), which supports the concept that ADM decreases water permeability by stimulating cAMP-independent pathways. U73122 significantly increased phosphorylation at serine 256 but did not change phosphorylation of AQP2 at serine 261 ( Figure 8), which suggests that the increase in ADM-attenuated water permeability by PLC inhibition may be related to the increase in AQP2 trafficking to the plasma membrane instead of a decrease in endocytosis of AQP2.
Given that both PKC and cGMP are downstream signals of PLC signaling pathways, it is likely that PKC and/or cGMP are involved in ADM-regulated water reabsorption. To investigate whether PKC regulates ADM-attenuated water permeability, the PKC inhibitor, rottlerin, was used to reverse the osmotic water permeability. Our data show that inhibition of PKC by rottlerin significantly increases ADM-attenuated water permeability (Figure 9), which indicates that ADM regulates water transport through PKC. Blocking PKC with rottlerin increased phosphorylation of AQP2 at serine 256 but did not alter phosphorylation of AQP2 at serine 261 ( Figure 10), which is consistent with the effect of inhibition of PLC with U73122. To determine whether cGMP changes ADM-mediated phosphorylation of AQP2, the nitric oxide synthase inhibitor, L-NAME was used to block the phosphorylation of AQP2 by ADM. Our results show that inhibition of cGMP does not change AQP2 phosphorylation at serine 256 and serine 261 (Figure 11), which suggests that ADM does not regulate water permeability through cGMP. Therefore, we verified that ADM reduces osmotic water permeability through a PLC-PKC signaling pathway. However, we need to point out that ADM may also involve other signaling pathways to regulate water reabsorption because the PLC-PKC signaling pathway does not change phosphorylation of AQP2 at serine 261. How ADM increases phosphorylation of AQP2 at serine 261 needs to be further investigated.
In conclusion, the present study shows that ADM and its receptors are expressed in rat inner medulla. ADM stimulates cAMP production, but ADM regulates water reabsorption through cAMP-independent pathways. ADM reduces water transport by activating the PLC-PKC signaling pathway. The decrease in water permeability appears to be a result of a decrease in AQP2 phosphorylation at serine 256, which may impede AQP2 trafficking to the plasma membrane. ADM is a potential therapeutic target for treatment of cardiovascular diseases associated with water imbalance.

Conflicts of Interest:
The authors declare no conflict of interest with the representation or interpretation of the reported research results.