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Am J Physiol Renal Physiol 293: F1858-F1864, 2007. First published October 3, 2007; doi:10.1152/ajprenal.00068.2007
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Vasopressin receptor subtype 2 activation increases cell proliferation in the renal medulla of AQP1 null mice

Qi Cai,1 Matthew R. McReynolds,1 Maggie Keck,1 Kevin A. Greer,2 James B. Hoying,1,2 and Heddwen L. Brooks1

1Department of Physiology, College of Medicine and 2Arizona Research Laboratories, University of Arizona, Tucson, Arizona

Submitted 12 February 2007 ; accepted in final form 24 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Aquaporin (AQP) 1 null mice have a defect in the renal concentrating gradient because of their inability to generate a hyperosmotic medullary interstitium. To determine the effect of vasopressin on renal medullary gene expression, in the absence of high local osmolarity, we infused 1-deamino-8-D-arginine vasopressin (dDAVP), a V2 receptor (V2R)-specific agonist, in AQP1 null mice for 7 days. cDNA microarray analysis was performed on the renal medullary tissue, and 5,140 genes of the possible 12,000 genes on the array were included in the analysis. In the renal medulla of AQP1 null mice, 245 transcripts were identified as increased by dDAVP infusion and 200 transcripts as decreased (1.5-fold or more). Quantitative real-time PCR measurements confirmed the increases seen for cyclin D1, early growth response gene 1, and activating transcription factor 3, genes associated with changes in cell cycle/growth. Changes in mRNA expression were correlated with changes in protein expression by semiquantitative immunoblotting; cyclin D1 and ATF3 were increased significantly in abundance following dDAVP infusion in the renal medulla of AQP1 null mice (161 and 461%, respectively). A significant increase in proliferation of medullary collecting ducts cells, following V2R activation, was identified by proliferating cell nuclear antigen immunohistochemistry; colocalization studies with AQP2 indicated that the increase in proliferation was primarily observed in principal cells of the inner medullary collecting duct (IMCD). V2R activation, via dDAVP, increased AQP2 and AQP3 protein abundance in the cortical collecting ducts of AQP1 null mice. However, V2R activation did not increase AQP2 protein abundance in the IMCD of AQP1 null mice.

cyclin D1; microarray; activating transcription factor 3

VASOPRESSIN, a peptide hormone, controls systemic osmolality through regulation of renal water excretion. Vasopressin mediates its physiological effects via the vasopressin type 2 receptor (V2R) to stimulate intracellular cAMP formation and activate protein kinase A. Aquaporin (AQP) 2 is the main target of vasopressin action; hence, collecting duct water permeability is increased via AQP2 insertion in the collecting duct membrane. V2Rs are expressed on the basolateral membrane of collecting duct cells, and activation mediates both rapid and long-term regulation of cell water, urea, and sodium permeability. Many studies have documented the long-term molecular regulation of AQP2 by vasopressin action on V2Rs in renal collecting duct cells. Infusion of vasopressin increases the water permeability of collecting ducts in vasopressin-deficient Brattleboro rats, and this effect correlates well with increased levels of AQP2 mRNA and protein (2, 7). Recent studies have also identified a role for hyperosmolality in the regulation of AQP2 expression in vivo and in vitro, independent of vasopressin (14, 31).

The presence of AQP1 water channels in thin descending limbs plays a vital role in countercurrent multiplication, since they allow rapid osmotic equilibration along the tubule. A study of AQP1 null mice demonstrated their failure to produce concentrated urine upon water deprivation (15). Data from several laboratories suggests that the primary renal defect in AQP1 null mice is the inability to generate a hyperosmotic medullary interstitium due to the loss of countercurrent multiplication (4, 24). This study uses AQP1 null mice as a tool to determine the specific effects of vasopressin on medullary gene expression, in the absence of high osmotic stress. Activation of the V2R was performed by infusing the AQP1 null mice with 1-deamino-8-D-arginine-vasopressin (dDAVP), a V2R specific agonist. We report here the microarray analysis of changes in medullary gene expression following 7 days of dDAVP infusion and the identification of a V2R-mediated increase in the expression of cell proliferation genes in the renal medulla. Follow-up studies were performed to examine the protein expression of cyclin D1, activating transcription factor 3 (ATF3), and proliferating cell nuclear antigen (PCNA), an indicator of cell proliferation, in control and dDAVP-treated AQP1 null mice. A significant increase in medullary collecting duct cell proliferation following dDAVP infusion in AQP1 null mice was confirmed, using immunohistochemistry. This increase in cell proliferation was not observed with V2R activation in wild-type mice. Thus we find that an increase in V2R activation, in the nonconcentrating medulla, increases cell proliferation mainly in the principal cells of the renal collecting duct.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Alzet microosmotic pumps (no. 1007D) were obtained from Durect (Cupertino, CA). The RNeasy Mini Kit (no. 74104), PCR purification kit (no. 28104), and SYBR Green reagents (no. 20414) were from Qiagen (Valencia, CA). The MessageAmp kit (no. 1750) and EndoFree RT kit (no. 1740) were from Ambion (Austin, TX). Polyvinylidene difluoride (PVDF) membrane (no. IPVH00010) was from Millipore (Billerica, MA). The BCA kit (no. 23227) was from Pierce (Rockford, IL). Mouse anti-PCNA (F2; no. sc-25280), mouse anti-cyclin D1 (DCS-6; no. sc-20044), and rabbit anti-ATF3 (C-19; no. sc-188) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-GRP78 (no. SPA-826) was from Stressgen Bioreagents (Victoria, BC, Canada). AQP2, AQP3, and AQP4 rabbit polyclonal antibodies were generously provided by Dr. M. A. Knepper (National Institutes of Health). Goat anti-rabbit IgG (no. 7074) and goat anti-mouse IgG (no. 7076) were from Cell Signaling Technology (Danvers, MA). The enhanced chemiluminiscence (ECL) Plus Western Blotting Detection system (no. RPN2132) was from Amersham Biosciences (Piscataway, NJ). Biotin-goat anti-rabbit IgG (no. 81-6140), biotin-rabbit anti-mouse IgG (no. 81-6740), horseradish peroxidase-streptavidin conjugate (no. 43-4323), and diaminobenzidine (DAB) substrate kit (no. 00-2014) were from Zymed Laboratories (S. San Francisco, CA). Fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit (no. 711-095-12) and Texas red-conjugated donkey anti-mouse (no. 715-075-150) were from Jackson ImmunoResearch (West Grove, PA). Vectashield (no. H-1000) was from Vector Laboratories (Burlingame, CA). Alexa Fluor 546 (no. A-20002) and Alexa Fluor 647 (no. A-20006) were from Molecular Probes (Eugene, OR).

Experimental animals. AQP1 null mice were generated by homologous recombination in embryonic stem cells as previously reported (15). The mice were bred and maintained in the animal facility of the University of Arizona Health Sciences Center under National Institutes of Health guidelines. Genotypes designated "+" for the wild-type allele and "–" for the targeted allele were determined by PCR analysis of genomic DNA isolated from tail biopsies. The mice used in the present study were 8 wk old and received regular food and water ad libitum. Osmotic mini pumps were loaded with dDAVP and subcutaneously implanted in mice under light anesthesia of isoflurane. The infusion rate of dDAVP was 0.5 ng/h for 7 days. Mouse urine was collected by allowing mice to urinate on parafilm, and urine osmolality was measured using a vapor pressure osmometer (model 5100B; Wescor).

RNA isolation, amplification, and cDNA purification. Full methodology for the RNA purification, amplification, and cDNA production has been previously published (17). Briefly, RNA samples isolated from individual mouse inner medullas were labeled C1–C3 for AQP1 null mice and E1–E3 for dDAVP-infused AQP1 null mice. RNA was amplified using the MessageAmp kit according to the manufacturer's protocols. Total RNA (3 µg) from each individual mouse was used as a template for each amplification reaction, and this gave a yield of ~50 µg of amplified RNA. Amplified RNA was then reverse transcribed to cDNA using the EndoFree RT kit according to the manufacturer's protocol. Amino allyl-modified cDNA was purified using PCR purification columns according to the manufacturer's protocol. The modified cDNA was labeled with Alexa dyes 546 Alexa Fluor and 647 Alexa Fluor via the free amine modification.

Microarray slide preparation, hybridization, and analysis. Microarrays for our study were prepared within the Genomic Research Laboratory at the University of Arizona using the National Institute on Aging mouse 15K clone set http://lgsun.grc.nia.nih.gov/cDNA/15k.html. Full methodology for the production of the microarrays and the hybridization protocols has been published previously (17). Labeled modified cDNA in hybridization buffer was loaded on a slide and set to hybridize at 47°C for a minimum of 16 h. After completion, a short wash was run in the hybridization station after which the slide was removed and dipped in 0.05x saline-sodium citrate to remove any residual nonhybridized cDNA. The slide was dried and analyzed using the arrayWORxe CCD-based microarray scanner from Applied Precision, capable of multichannel fluorescence scanning. Microarray data were reduced and evaluated by CARMA as previously described (9, 17); the results were submitted to the Gene Expression Omnibus.

Real-time quantitative PCR. Real-time quantitative PCR was carried out using the Rotor-Gene RG-3000 (Corbett Research) sequence detection system and SYBR Green reagents. Primers were designed using Primer3 software (23) and are listed in supplemental data B along with the gene accession number for the target gene (Supplemental data for this paper can be found at the American Journal of Physiology: Renal Physiology web site.). Total or amplified RNA (3 µg) was reverse transcribed with the Endofree RT kit, according to the manufacturer's protocol. The cDNA was diluted to 8 ng/µl, and the PCR reaction mixture contained 5 µl of Sybr master mix, 0.4 µl 25 mM MgCl2, 0.6 µl RNase-free water, 5 pmol of forward and reverse primers, and 16 ng cDNA in a volume of 10 µl. Each reaction was performed in triplicate at 95°C for 15 min and then at 95°C for 15 s, 58°C for 15 s, and 20 s at 72°C for 40 cycles. This was followed by a melt cycle that consisted of a stepwise increase in temperature from 72 to 99°C. A single predominant peak was observed in the dissociation curve of each gene, supporting the specificity of the PCR product. Threshold values were set within the exponential phase of the PCR and were used to calculate the expression levels of the genes of interest before being normalized to endogenous cellular dynactin RNA. The level of dynactin RNA was measured in parallel samples using dynactin-specific primers.

Protein sample preparation, SDS-PAGE, and Western blot. Kidneys were dissected into inner medullas and cortexes and homogenized in ice-cold isolation solution (250 mM sucrose and 10 mM triethanolamine, pH 7.6, containing 1 mg/ml leupeptin and 0.1 mg/ml phenylmethylsulfonyl fluoride) using a tissue homogenizer (Omni 1000 fitted with a microsawtooth generator) at maximum speed for three 15-s intervals. Total protein concentrations were measured using the BCA kit, and the samples were solubilized in Laemmli sample buffer at 60°C for 15 min. Proteins were separated on 10 or 12% SDS-PAGE gels and transferred to a PVDF membrane. Membranes were blocked for 1 h at room temperature with 5% nonfat dry milk and then incubated overnight at 4°C with the primary antibodies, followed by incubation of horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature (1:2,000 dilution). Horseradish peroxidase was visualized using the ECL plus Western Blotting Detection System. Band densities were determined by the BioIamging System (UVP, Upland, CA).

Immunostaining. For immunohistochemistry staining, mouse kidneys were fixed in 4% paraformaldehyde overnight. Sections (4 µm) prepared by the pathology laboratory at the University of Arizona were deparaffinized with xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was quenched with 0.3% (vol/vol) hydrogen peroxide in absolute methanol for 30 min. Antigen was retrieved by heating the sections in citrate buffer (pH 6.0) in a microwave oven for 10 min. Nonspecific binding was blocked in 2% BSA. Sections were then incubated overnight at 4°C with primary antibodies, followed by biotin-goat anti-rabbit IgG or biotin-rabbit anti-mouse IgG (1:200 dilution) for 30 min at 37°C and horseradish peroxidase-streptavidin conjugate (1:200 dilution) for 30 min at 37°C. Labeling was visualized with chromogen DAB. Finally, the slides were counterstained with hematoxylin. For immunofluorescent staining, the protocol used is the same as that used in immunohistochemistry except for the secondary antibodies, which are FITC-conjugated donkey anti-rabbit or Texas red-conjugated donkey anti-mouse. The slides were mounted with antifade medium (Vectashield).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of dDAVP infusion on urine osmolality in wild-type and AQP1 null mice. Short-term increases in vasopressin, either via water restriction (48 h) or dDAVP (0.4 µg/kg, single injection), have previously been shown to have no effect on urine osmolality in AQP1 null mice (15). To investigate the role of long-term vasopressin receptor activation on the urine concentrating ability in AQP1 null mice, the vasopressin analog dDAVP was delivered at 0.5 ng/h for 7 days. In wild-type mice, dDAVP infusion for 7 days significantly increased urine osmolality (dDAVP infusion 2,847 ± 87 mosmol/kgH2O compared with 1,644 ± 190 mosmol/kgH2O in control mice), as shown in Fig. 1. In contrast, AQP1 null mice demonstrated a small increase in urine osmolality following 7 days of dDAVP infusion (dDAVP infusion: AQP1 null mice 893 ± 62 mosmol/kgH2O compared with control AQP1 null mice 581 ± 26 mosmol/kgH2O, P < 0.05), which, although significant compared with control mice, was only an increase of 300 mosmmol/kgH2O (Fig. 1).


Figure 1
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  		Fig. 1. Effect of 1-deamino-8-D-arginine vasopressin (dDAVP) infusion on urine osmolality in aquaporin (AQP) 1 null mice. AQP1 null mice and wild-type mice were infused with dDAVP at 0.5 ng/h for 7 days. Urine was collected, and osmolality was measured using a vapor pressure osmometer. Data are presented as means ± SE; n = 3 experiments. *Significant difference between control and dDAVP-infused mice (t-test; P < 0.05).

 
Microarray analysis. Total RNA was isolated from the inner medulla of AQP1 null mice (samples C1, C2, and C3) and dDAVP-infused AQP1 null mice (samples E1, E2, and E3) for microarray analysis of gene expression using our previously published methods (3, 17). Total RNA from each sample was then amplified before being labeled in the cDNA reaction, and each sample from an individual mouse was hybridized to an array four times. Data were analyzed and transformed as previously described (3, 17). The ANOVA was performed on a gene-by-gene basis and was limited to genes that were measured confidently on a minimum of three out of the four hybridizations, for at least one sample. In this study, 5,140 genes out of the possible ~12,000 genes on the array were included in the ANOVA. Genes were considered to be significantly differentially expressed if the ANOVA P value was <0.05. Genes that exhibited small changes in gene expression but were identified as significant because of unusually small variance were excluded based on a cut off of ≥1.5-fold up- or downregulation between the AQP1 null and dDAVP-infused AQP1 null mice. Four hundred forty-five genes were in this selected group, 245 were significantly increased and 200 were significantly decreased following dDAVP infusion. An output file showing individual measurements, gene name (if known), and links to the NIA and Genbank databases was generated (see supplemental data A; Supplemental data for this paper can be found at the American Journal of Physiology: Renal Physiology web site.).

dDAVP increases growth-regulated genes and induces cell proliferation in inner medulla of AQP1 null mice. Microarray data identified cyclin D1, early growth response gene 1 (Egr-1), and ATF3 mRNA as significantly increased in the medulla of AQP1 null mice 7 days after dDAVP infusion. The elevation of all three genes was confirmed by real-time PCR analysis (Fig. 2A). To confirm that the increase in mRNA expression does not occur in the normal concentrating medulla, mRNA expression levels were determined in wild-type mice following dDAVP infusion for 7 days. As shown in Fig. 2B, cyclin D1, Egr-1, and ATF3 mRNA did not increase in the medulla of wild-type mice following 7 days of dDAVP infusion. In fact, as shown in Fig. 2B, and similar to our previous studies in water-restricted mice (3), cyclin D1 mRNA expression is reduced in the medulla of wild-type mice following dDAVP infusion.


Figure 2
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  		Fig. 2. Effect of dDAVP infusion on mRNA expression of growth-related genes in renal inner medulla of wild-type and AQP1 null mice. SYBR Green I real-time PCR assay validation for growth-related genes. The data shown are the mean relative fold change in the renal medullas of control and dDAVP-infused AQP1 null (AQP1KO) mice (A) and control and dDAVP-infused wild-type (WT) mice (B). *Significant difference between control and dDAVP-infused mice (t-test; P < 0.05). Mice were infused with dDAVP at 0.5 ng/h for 7 days. Assays for dynactin were run in parallel on each sample for subsequent normalization of the data. Egr-1, early growth response gene 1; ATF3, activating transcription factor 3.

 
To confirm that mRNA changes correlated with changes in protein expression, Western blot analysis was performed on renal medullary tissue from a different set of AQP1 knockout mice following 7 days of dDAVP infusion. As shown in Fig. 3, cyclin D1 and ATF3 protein expression was significantly increased in the renal medulla of AQP1 null mice following 7 days of dDAVP infusion. It was not possible to measure the protein expression of Egr-1 at this time, since available antibodies did not work well in kidney tissues. ATF3 is a multifunctional transcription factor that can be involved in cell growth (1, 27). Cyclin D1 is a cell cycle regulatory protein whose expression increases in the G2 phase and is maintained through mitosis and the G1 phase. High levels of cyclin D1 in the G1 phase promote cells to enter the S phase (26). Follow-up studies were performed to determine if this change in expression of cyclin D1, ATF3, and Egr-1, following dDAVP infusion, was accompanied by an increase in medullary cell proliferation.


Figure 3
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  		Fig. 3. dDAVP infusion increase the protein expression of cyclin D1 and ATF3 in the renal inner medulla of AQP1 null mice. AQP1 null mice were infused with dDAVP at 0.5 ng/h for 7 days. Protein abundance was analyzed by Western blot. Each lane represents a homogenate from an individual mouse. Densitometry values are normalized to a control value of 100 to facilitate comparison. *Significant difference between AQP1 null mice and dDAVP-infused AQP1 null mice (t-test, P < 0.05).

 
PCNA is a marker of cell proliferation. As shown in Fig. 4, there was a significant increase in PCNA protein expression in the renal medullas of AQP1 null mice after dDAVP infusion (dDAVP infused 355 ± 60.6% compared with 100 ± 14.7% in control AQP1 null mice, P < 0.05). In contrast, there was no increase in PCNA staining in the renal medulla of wild-type mice after dDAVP infusion. To identify which cell type of the renal medulla was positive for PCNA, we used immunohistochemistry and immunofluorescence. As shown in Fig. 5, the majority of PCNA-positive cells was located in the initial part of the inner medullary collecting ducts (IMCD). There were no PCNA-positive cells in the terminal IMCDs.


Figure 4
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  		Fig. 4. dDAVP infusion increases proliferating cell nuclear antigen (PCNA) protein expression in the renal medulla of AQP1 null mice but not in wild-type mice. AQP1 null mice were infused with dDAVP at 0.5 ng/h for 7 days. Immunoblots examining PCNA protein abundance in inner medulla homogenates of control and AQP1KO mice (A) and control and WT mice (B). Each lane represents a homogenate from an individual mouse. Densitometry values are normalized to a control value of 100 to facilitate comparison. *Significant difference between AQP1 null mice and dDAVP-infused AQP1 null mice (t-test, P < 0.05).

 

Figure 5
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  		Fig. 5. Localization of dDAVP-induced cell proliferation to collecting ducts in the initial renal medulla of AQP1 null mice. PCNA immunoperoxidase labeling in paraffin-embedded mouse kidney sections from control and AQP1KO mice. A: renal medullas of control and dDAVP-infused AQP1 null mice; arrow demonstrates region of PCNA positive staining in medulla (magnification x40). B: immunoperoxidase labeling in AQP1KO mice demonstrating dDAVP-induced cell proliferation is localized to collecting duct tubules in renal medulla (magnification x100).

 
AQP2 is expressed exclusively in the collecting duct cells of the kidney, and it can be used to identify principal cells of the collecting duct. AQP2 and PCNA dual labeling was performed and, as shown in Fig. 6, proliferating cells were positive for AQP2, suggesting that it is primarily principal cells that are proliferating in the renal medulla of AQP1 null mice following dDAVP infusion. However, not all PCNA-positive cells colocalized with AQP2, indicating that there are either some undifferentiated cells or nonprincipal cells also proliferating.


Figure 6
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  		Fig. 6. Immunolocalization of PCNA to principal cells of renal collecting ducts. AQP1 null mice were infused with dDAVP at 0.5 ng/h for 7 days. Immunofluorescent localization of PCNA in red (A) and AQP2 in green (B) to principal cells of collecting duct tubules in the mouse medulla. C: merged image. Arrows identify AQP2 and PCNA positive principal cells (magnification x400).

 
A recent in vivo study observed an increase in medullary cell proliferation in lithium-treated rats, with cell proliferation identified as occurring primarily in principal cells (5). p27/Kip, a cyclin-dependent kinase inhibitor, was identified via microarray analysis as decreased in expression in the medulla of lithium-treated rats; thus, the authors suggested that the increase in cell proliferation may be because of the loss of p27/Kip inhibition (5, 22). Using Western blot analysis, we demonstrate that the protein expression of p27/KIP is also decreased in the renal medulla of AQP1 null mice following dDAVP infusion, as shown in Fig. 7.


Figure 7
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  		Fig. 7. dDAVP infusion reduces the expression of p27/KIP in renal medulla of AQP1 null mice. AQP1 null mice were infused with dDAVP at 0.5 ng/h for 7 days. p27/KIP protein abundance was analyzed by Western blot. Each lane represents a homogenate from an individual mouse. Densitometry values are normalized to a control value of 100 to facilitate comparison. *Significant difference between AQP1 null mice and dDAVP-infused AQP1 null mice (t-test, P < 0.05).

 
Medullary AQP regulation is altered in AQP1 null mice. Many studies have demonstrated that V2R activation, via dDAVP infusion, increases the expression of both AQP2 and AQP3 in rat cortical collecting ducts and increases AQP2 expression in rat IMCD cells (28). To determine if the long-term action of dDAVP on renal AQP expression in the AQP1 null mice was intact, we used Western blot analysis to measure the protein expression of AQP2 and AQP3 in both renal cortical and medullary collecting ducts. Following 7 days of dDAVP infusion, AQP2 and AQP3 protein expression were significantly increased in the cortex of AQP1 null mice as shown in Fig. 8, A and B (AQP2 and AQP3 protein increased to 163 ± 16.8 and 240 ± 26.9%, respectively, compared with control, 100%, P < 0.05). In contrast, AQP2 and AQP3 protein expression did not increase in the medullary collecting ducts of AQP1 null mice following dDAVP infusion, shown in Fig. 8, C and D.


Figure 8
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  		Fig. 8. Effect of dDAVP infusion on the protein expression of AQP2 and AQP3 in cortex and medulla of AQP1 null mice. AQP1 null mice were infused with dDAVP at 0.5 ng/h for 7 days. Protein samples were separately prepared from renal cortex (A) and inner medulla (C) for Western blot analysis. Each lane represents a homogenate from an individual mouse. B and D: densitometry analysis of A and C, respectively. Densitometry values are normalized to a control value of 100 to facilitate comparison. *Significant difference between AQP1 null mice and dDAVP-infused AQP1 null mice (t-test, P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, in AQP1 null mice, microarray analysis was used to determine the gene expression changes that occur downstream of V2R activation. We identified the upregulation of several genes associated with cell cycle regulation and confirmed the microarray data at both the RNA and protein levels. We also demonstrated that dDAVP infusion increased cell proliferation in the renal medulla of the AQP1 null mice. Because previous studies have demonstrated that medullary collecting duct cells in the mature kidney divide at a very slow rate (33), we used colocalization studies to identify which cell type in the medulla was proliferating. Proliferating cells in the medulla were identified as mainly AQP2-expressing principal cells.

Microarray analysis identified Egr-1, ATF3, and cyclin D1 as increased in expression in the medulla of AQP1 null mice after V2R activation with dDAVP. Egr-1 is a zinc finger transcription factor and can be rapidly and transiently induced by growth factors and other extracellular signals. It was reported that a cAMP response element exists in the promoter region of Egr-1 and can mediate Egr-1 transcription (12, 13). Once activated, Egr-1 can regulate the expression of several genes, one of which is cyclin D1 (10, 32) and thus can be an important mediator of cell proliferation (19, 29). In the present study, dDAVP increased cyclin D1 mRNA and protein expression in the renal medulla of AQP1 null mice. Cyclin D1, a cell cycle regulatory protein, is known to promote cell entry into and completion of S phase via its association with a cyclin-dependent kinase and the subsequent phosphorylation of retinoblastoma protein. Thus cell-cycle progression depends on the activation of cyclins and cyclin-dependent kinases. However, the cell cycle is tightly controlled by cyclin-cyclin dependent kinase complexes and their inhibitors such as p27/Kip. The p27/Kip is a straightforward inhibitor of G1 phase progression. In muscle satellite cells, suppressing p27/Kip expression allowed cells to proliferate, and overexpression of p27/Kip inhibited cell proliferation (25). In many tumor cells, p27/Kip expression is decreased (20). In the present study, dDAVP increases cyclin D1 and decreases p27/KIP protein expression in the medulla of AQP1 null mice; these changes are accompanied by an increase in cell proliferation. Thus our observations suggest that regulation of both cyclin D1 and p27/kip may be associated with the increased cell proliferation induced by dDAVP.

V2R activation increases intracellular cAMP in collecting duct principal cells; thus, it is plausible that both Egr-1 and cyclin D1 are involved in a cell proliferation pathway in the inner medulla of AQP1 null mice following dDAVP infusion. It remains to be determined whether they act in concert, each with a specific role, or whether both act in a similar way such that the induction of either gene is sufficient to induce proliferation. Further in vitro experiments are required to delineate how these genes are involved in the regulation of V2R-activated medullary cell proliferation.

The role of cAMP in renal cell proliferation is complex. It is well known that cAMP is involved in the proliferation pathway of renal collecting duct cells in both humans and rodents with polycystic kidney disease (PKD). Similar to the AQP1 null mice, PKD mice have a defect in urinary concentrating ability. Recent studies in PKD mice demonstrated that cAMP levels were increased significantly in the PKD kidney compared with normal mice (30). Blockade of the V2R in these mice, using a specific nonpeptide antagonist OPC-31260, inhibited disease development, including cell proliferation and cyst formation, and reduced renal cAMP levels (30).

Recently, it has also been reported that a 4-wk lithium treatment is able to induce collecting duct cell proliferation in the renal medulla of normal rats; this was also accompanied by a decrease in p27/Kip protein expression (5, 22). Similar to our study, proliferation was observed mainly in principal cells. It is well known that prolonged exposure to lithium results in reversible nephrogenic diabetes insipidus by reducing AQP2 expression over time and diminishing the interstitial osmolality of the medulla because of the depletion of urea (6). However, lithium is itself a mitogen in some cell types (11, 21).

These studies demonstrate that renal medullary collecting duct cells are capable of proliferating, although whether a reduced medullary osmolality is required for collecting duct cells to respond to a mitogenic signal, be that vasopressin (via cAMP) or lithium, is currently unknown. In vitro, hyperosmolality has been demonstrated to inhibit cell proliferation in a renal medullary collecting duct cell line, mIMCD3 (18), suggesting that the loss of medullary osmolality in the AQP1 null mice may be a key factor in the increase in cell proliferation, following V2R activation observed here.

Osmolality has been demonstrated to play a role in the regulation of AQP2 expression in the renal collecting duct (14) (31). In a recent study, Brattleboro rats were either given hypertonic saline instead of drinking water, or diabetes mellitus was induced; AQP2 protein expression was increased in the medullary collecting duct in both studies (14). Because Brattleboro rats do not have circulating vasopressin, this increase was attributed to the increased extracellular hyperosmolality, and thus medullary osmolality (14). In this study, we demonstrated that V2R activation was sufficient to increase AQP2 expression in the cortical collecting duct of AQP1 null mice. However, in the medullary collecting duct, no change in AQP2 expression was observed after dDAVP infusion. In lithium-treated animals, it is well documented that AQP2 protein expression is reduced in renal collecting duct cells (16, 16); in contrast, AQP2 expression is increased in the collecting duct cells of PKD mice (8). The loss of high osmolality in the AQP1 null mice may explain why dDAVP infusion in our current study did not increase AQP2 expression. However, if this is the case, future studies to increase local osmolality in these mice, using hypertonic saline as drinking water, may shed light on this observation. In summary, we have identified an increase in medullary collecting duct cell proliferation, accompanied by changes in expression of cell cycle genes, in the AQP1 null mice following V2R activation by dDAVP. In addition, no V2R-mediated increase in AQP2 expression occurred in the collecting ducts of these AQP1 null animals, suggesting that medullary cell proliferation causes changes in the regulation of AQP2 expression and hence could affect collecting duct water permeability.

The role of high circulating vasopressin, such as found in heart failure and diabetes, is unknown in the renal medulla when the renal concentrating mechanism is reduced. In a clinical setting, it is common for renal medullary osmolality to be reduced; the diuretic furosemide rapidly reduces the countercurrent multiplication mechanism via its action on the thick ascending limb and "washes out" the renal medullary gradient. Our findings in this study suggest that vasopressin receptor-activated pathways are altered in this situation of low extracellular hyperosmolality and provide compelling rationale for conducting further mechanistic studies related to vasopressin receptor signaling in clinical models of renal disorders.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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This work was funded by a grant from the University of Arizona Foundation (H. L. Brooks), by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-064706 (H. L. Brooks), and by the American Physiological Society's Physiological Genomics Fellowship (Q. Cai).


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Brooks, Dept. of Physiology, College of Medicine, 1501 N. Campbell Ave., Univ. of Arizona, Tucson, AZ 85724-5051 (e-mail: brooksh{at}email.arizona.edu)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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