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Am J Physiol Renal Physiol 295: F1177-F1190, 2008. First published August 20, 2008; doi:10.1152/ajprenal.00465.2007
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V2 vasopressin receptor deficiency causes changes in expression and function of renal and hypothalamic components involved in electrolyte and water homeostasis

Nicole Schliebe,1,* Rainer Strotmann,1,* Kathy Busse,1 Doreen Mitschke,1 Heike Biebermann,2 Lutz Schomburg,3 Josef Köhrle,3 Jörg Bär,1 Holger Römpler,1 Jürgen Wess,4 Torsten Schöneberg,1 and Katrin Sangkuhl1

1Institute of Biochemistry, Molecular Biochemistry, Medical Faculty, University of Leipzig, Leipzig; 2Institute of Experimental Pediatric Endocrinology and 3Institute of Experimental Endocrinology, Charité, Medical Faculty, Humboldt University Berlin, Berlin, Germany; and 4Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland

Submitted 8 October 2007 ; accepted in final form 23 July 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Polyuria, hypernatremia, and hypovolemia are the major clinical signs of inherited nephrogenic diabetes insipidus (NDI). Hypernatremia is commonly considered a secondary sign caused by the net loss of water due to insufficient insertion of aquaporin-2 water channels into the apical membrane of the collecting duct cells. In the present study, we employed transcriptome-wide expression analysis to study gene expression in V2 vasopressin receptor (Avpr2)-deficient mice, an animal model for X-linked NDI. Gene expression changes in NDI mice indicate increased proximal tubular sodium reabsorption. Expression of several key genes including Na+-K+-ATPase and carbonic anhydrases was increased at the mRNA levels and accompanied by enhanced enzyme activities. In addition, altered expression was also observed for components of the eicosanoid and thyroid hormone pathways, including cyclooxygenases and deiodinases, in both kidney and hypothalamus. These effects are likely to contribute to the clinical NDI phenotype. Finally, our data highlight the involvement of the renin-angiotensin-aldosterone system in NDI pathophysiology and provide clues to explain the effectiveness of diuretics and indomethacin in the treatment of NDI.

vasopressin receptor; diabetes insipidus; G protein-coupled receptor; signal transduction; hypernatremia

THE NEUROHYPOPHYSEAL PEPTIDE arginine vasopressin (AVP) is an essential hormone in mammals that regulates the body's water and electrolyte homeostasis. AVP mediates its renal actions through activation of the tubular V2 vasopressin receptor (Avpr2). Avpr2 stimulation activates the Gs protein/adenylyl cyclase signaling cascade and eventually results in the insertion of the water channel protein aquaporin-2 (AQP2) into the apical plasma membrane of the collecting duct cells (42). The major physiological importance of the AVP regulatory system becomes apparent when key components are defective. AVP deficiency results in central diabetes insipidus (CDI), whereas inactivating mutations in Avpr2 or AQP2 genes lead to inherited forms of nephrogenic diabetes insipidus (NDI). In NDI, the renal concentrating capability is impaired and the kidney produces large volumes of hypotonic urine, a situation that may lead to severe dehydration and electrolyte imbalance, in particular hypernatremia and hyperchloremia (29). Mechanistically, hypernatremia is believed to be the result of net water depletion. This assumption is supported by effective treatment of NDI patients with abundant water intake. Current therapy of NDI patients further includes a low-sodium diet and thiazide diuretics, often in combination with the potassium-sparing diuretic amiloride.

Thiazide diuretics [e.g., hydrochlorothiazide (HCTZ)] paradoxically decrease urine volume and increase urine osmolality in NDI patients (21). A recent study (40) showed upregulation of AQP2 and a number of distal renal Na+ transporters, such as the thiazide-sensitive NaCl cotransporter (NCC) and the epithelial sodium channel (ENaC) in response to HCTZ treatment. The authors proposed that this effect accounts for the antidiuretic action of HCTZ in lithium-induced NDI (40). They suggested that upregulation of NCC and ENaC decreases luminal osmolality in the cortical connecting tubule and the collecting duct, thus increasing the driving force for water reabsorption (40).

In the treatment of NDI, thiazides are often combined with indomethacin, a nonsteroidal anti-inflammatory drug (NSAID). Indomethacin, a nonselective cyclooxygenase type 1 and 2 (COX1/2) inhibitor, decreases urine volume, free water clearance, and increases urinary osmolality without affecting renal or glomerular blood flow (94). The effect of indomethacin on urine volume is most likely due to its inhibition of prostaglandin (PG)-dependent water filtration. However, the therapeutic actions of thiazides and NSAID in NDI are complex and still not well understood.

Detailed information on the adaptive changes in the kidney and other parts of the renal-hypothalamic regulatory system in NDI patients is crucial for a better understanding of the therapeutic actions of thiazides and NSAID. A number of expression profiling studies have been performed in several (patho)physiological circumstances that result in electrolyte imbalance, including lithium-induced NDI (12, 14, 58, 71). However, little is known about adaptive changes in the kidneys of NDI patients lacking functional Avpr2 (X-linked NDI). In the present study, we analyzed adaptive responses in an Avpr2-deficient mouse model (99) using both transcriptome-wide microarray-based expression studies as well as functional characterization of selected components in kidney and hypothalamus. The mouse strain was generated using gene-targeting technology to introduce a premature stop mutation (E242X) into the Avpr2 gene that is located on the X chromosome. Hemizygous male E242X mice (NDI mice) are not capable of adequate urine concentration and die during the first neonatal week, probably due to hypernatremic dehydration (99). We have preferred this model to an animal model with induced or partial NDI phenotype to properly reflect the untreated life-threatening NDI pathophysiology. In NDI mice, like in human patients, the loss of gene function and the resulting pathophysiological changes are present at the very beginning of life. In contrast to human NDI patients who are typically treated by adequate water substitution, NDI mouse pups die during the first postnatal week because they are not individually nursed. We found that the expression of several genes involved in sodium and water reabsorption was upregulated in the kidneys of 3-day-old male Avpr2-deficient mice. This included AQP1, carbonic anhydrases (CAR), Na+-K+-ATPase, several Na+- and HCO3– transporters and transporter-modulating components. Interestingly, a considerable number of these genes is known to be regulated by aldosterone. Compared with lithium-induced NDI (41, 47), our analysis indicates a characteristic gene expression pattern for the X-linked form of NDI. In-depth expression analysis of components of the prostanoid and leukotriene metabolism revealed upregulation of COX2 expression in both, kidney and hypothalamus but also tissue-specific changes in the downstream eicosanoid pathways. Our data provide new insights into the pathophysiology of X-linked NDI and may explain the effectiveness of thiazides and indomethacin in the treatment of this disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mouse strain containing the E242X mutation and mouse genotyping. The generation and initial characterization of the mouse strain containing the E242X nonsense mutation has been reported previously (99). Briefly, the murine Avpr2 gene was inactivated in ES cells by introducing the E242X nonsense mutation into the Avpr2 coding region. The E242X mutation generated a novel NheI site that was used for genotyping (see below). To remove the loxP-flanked neo selection cassette from the genome of the Avpr2 mutant mice, homozygous male EIIa-cre mice were bred with female heterozygous E242X mutant mice. The resulting E242X mutant mice were backcrossed for 12 generations onto the 129/Sv mouse background. Animals were maintained in a controlled animal facility with 23°C room temperature, 60% humidity, and a 12:12-h light-dark cycle. All animal experiments were conducted in accordance with accepted standards of humane animal care and approved by the respective regional government agency of Saxony.

Mice were genotyped by PCR analysis of mouse-tail DNA. The two PCR primers used flanked the newly created NheI/E242X mutation: 5'-ATCTGCCGCCCTATGCTGGCATAC-3'/5'-CACAATCACTAGTGTCA CCTCAC-3' (94°C for 1 min/ 62°C for 1 min/ 72°C for 2 min, 35 cycles). The resulting PCR products were digested with NheI and separated on 2% agarose gels (note that only the fragment containing the E242X mutation contains a NheI cleavage site). For sex determination of the 3-day-old pups, a primer pair amplifying 450 bp of a Y chromosome-specific gene (SRY) was used: 5'-TCTTAAACTCTGAAGAAGAGAC-3'/5'-GTCTTGCCTGTAT GTGATGG-3' (94°C for 1 min/55°C for 1 min/72°C for 2 min, 35 cycles).

RNA isolation and microarray expression analysis. Kidneys and hypothalamus were removed from wild-type (WT) and NDI pups at postnatal day 3 and from 3-mo-old female mice. Total RNA was extracted from the tissues using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. Total RNA was further purified with RNeasy kits (Qiagen, Hilden, Germany) according to the RNA clean-up protocol. For microarray analysis, RNA integrity and concentration were quantified on an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA) using the RNA 6.000 LabChip Kit (Agilent Technologies) according to the manufacturer's instructions.

Microarray expression analysis of kidney and hypothalamus from three WT and three NDI mice, respectively, was performed at the IZKF Leipzig microarray core facility (IZKF Leipzig, Dr. K. Krohn). Ten micrograms of total RNA were reverse transcribed (SuperScript II, Invitrogen, Karlsruhe, Germany) using an oligo(dT) primer containing a T7 RNA polymerase promoter site (Genset, Paris, France). The cDNA was purified by phenol-chloroform extraction and subjected to in vitro transcription using the ENZO BioArray RNA transcript labeling kit (Affymetrix). Unincorporated nucleotides were removed using the RNeasy kit. The cRNA was fragmented and hybridized to GeneChip Mouse Genome Arrays 430A 2.0 (Affymetrix, Santa Clara, CA). Processing of the probe arrays was performed according to the manufacturer's instructions.

The expression data were normalized with the MAS5 and robust multiarray analysis (RMA) algorithms (36) using the R software package (http://www.r-project.org/). The annotation of the probe sets was obtained from Affymetrix. After filtering for transcripts that were detected as present in at least two samples of the same group, statistical testing of the log2-transformed data was done using a two-sided Student's t-test. For gene ontology profiling of MAS5-processed microarray data, probe sets with P ≤ 0.01 were subjected to the Onto Express algorithm (25). The settings were hypergeometric distribution, fdr correction. For gene set enrichment analysis, RMA- and MAS5-processed expression data were subjected to gene set enrichment analysis (GSEA) (84) using the software tool provided at http://www.broad.mit.edu/gsea/. Gene sets were derived from the Collecting Duct Database provided by the NIH (50), the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (38), the Molecular Signatures Database (MSigDB2.0) (85), and from the Mouse Genome 430A annotation file (Affymetrix) filtered for specific keywords within the gene ontology molecular function or gene title fields. GSEA parameters were 500 phenotype permutations with the datasets collapsed to gene symbols, weighted enrichment with signal-to-noise metric.

Preparation of cDNA and quantification by real-time PCR. For quantitative real-time PCR analysis (qPCR), total RNA was reverse transcribed (Superscript II, Invitrogen) with oligo(dT) primer. cDNA from 187.5 ng total RNA was subjected to qPCR using Platinum-SYBR Green qPCR Supermix (Invitrogen), 0.6 µM forward and reverse primers, and 100 nM ROX (5-carboxy-X-rhodamine, passive reference dye). If possible, oligonucleotide primers (supplemental Table S1; all supplementary material in this article is available at the journal web site) were designed with the Primer3 software (73) to flank intron sequences. PCR was performed in an MX 3000P instrument (Stratagene, La Jolla, CA) using the following protocol: 2 min 50°C, 2 min 95°C, and 50 cycles of 15 s 95°C, 30 s 60°C. A product melting curve was recorded to confirm the presence of a single amplicon. The correct amplicon size and identity were confirmed by agarose gel electrophoresis and restriction enzyme cleavage or sequencing. Standard curves with serial dilutions of cDNA were generated for each primer pair to assert linear amplification. Threshold cycle (cT) values were set within the exponential phase of the PCR. After normalization to β2-microglobulin, {Delta}cT values were used to calculate the relative expression levels (56). Gene regulation was statistically evaluated by subjecting the {Delta}{Delta}cT values derived from matched littermate samples to a two-sided Student's t-test, assuming equal variances. Gene regulation ratios are given as 2–{Delta}{Delta}cT values.

Western blot analysis of AQP1 expression. Both kidneys of 3-day-old WT or NDI mice were homogenized in lysis buffer (0.3 M sucrose, 25 mM imidazole, pH 7.2, 1 mM EDTA, 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 4,000 g for 15 min at 4°C, and the protein content of the supernatant was determined using Bradford reagent (Bio-Rad, Hercules, CA).

Total protein (150 and 75 µg) was subjected to 15% SDS-PAGE and electroblotted onto nitrocellulose membranes (Hybond-C Extra, Amersham, Piscataway, NJ). After being blocked with 5% nonfat dry milk in Tris-buffered saline (TBS; 7.7 mM Tris·HCl, pH 7.5, 150 mM NaCl) for 1 h at room temperature, the blots were incubated with anti-AQP1 antibody (Abcam, Cambridgeshire, UK) at a dilution of 1:500 in TBS with 2.5% nonfat dry milk for 1 h at room temperature. Membranes were extensively washed in TBS with 0.1% Tween 20 (TBST) and incubated with horseradish peroxidase-linked anti-mouse secondary antibody (Sigma, St. Louis, MO) diluted 1:1,000 in TBST containing 2.5% nonfat dry milk for 1 h at room temperature. After a wash at the conditions given above, immunostaining was detected using ECL Reagent (Amersham) and a chemiluminescence imaging system (DC Scientific). Band densitometry was done using Scion Image software (Scion, Frederick, MD).

Determination of enzyme activity: Na+-K+-ATPase. Total renal ATPase activity was determined by the quantification of inorganic phosphate release by ATP hydrolysis (28). Specific Na+-K+-ATPase activity was determined as the ouabain-sensitive fraction. In brief, both kidneys of 3-day-old pups or one-half kidney of adult animals were homogenized in homogenization buffer (see above) and plasma membranes were separated by ultracentrifugation (1 h, 100,000 g, 4°C). For further purification, the membrane fractions were resuspended at 1–1.5 mg/ml total protein in 300 mM sucrose, 25 mM imidazole, pH 7.5, 1 mM EDTA, and 0.055% SDS and incubated for 30 min at room temperature as described (37). The samples were centrifuged at 300,000 g for 90 min at 4°C in a discontinuous sucrose density gradient [29.4, 15, and 10% sucrose (wt/vol) in 25 mM imidazole, pH 7.5, 1 mM EDTA], and the pellets were resuspended in 25 mM imidazole, pH 7.5, containing 1 mM EDTA. For the determination of enzymatic activity, 10 µg of membrane protein was preincubated in 50 mM Tris·HCl, 4 mM MgCl2, 100 mM NaCl, 0.5 mM EDTA, 0.1% BSA, pH 7.0, with or without 6.25 mM ouabain (Sigma) in a final volume of 400 µl for 20 min at 37°C. The reaction was started by adding 100 µl of 10 mM ATP. Aliquots (100 µl) were taken from the reaction mixture at several time points, and the reaction was stopped by adding 50 µl of ice-cold trichloroacetic acid (12% wt/vol). Ten microliters of this mixture were transferred to 200 µl of dye reagent (0.034% Malachite green, 1.05% ammonium molybdate, 0.01% Tween 20, 1 M HCl) and the OD635nm was measured after 3 min. The phosphate release was calculated using a KH2PO4 standard curve. Aliquots of membrane protein preparations were used to determine the protein concentration with a standard Bradford assay. Ouabain-sensitive Na+-K+-ATPase activity was calculated as the difference in ATPase activities in the absence and presence of ouabain.

CAR. Both kidneys of 3-day-old pups or one-half kidneys of 3-mo-old mice were homogenized as described above, and the homogenates were diluted 1:1,000 with 25 mM imidazole, pH 7.0, 0.5 mM 4-nitrophenol. The CAR activity was determined at 4°C by stop-flow measurements in a SLM-Aminco DW 2000 spectrophotometer equipped with a rapid mixing attachment. The protons generated by the enzyme reaction were determined at 400 nm with 4-nitrophenol as the pH indicator. The reaction was started by mixing equal volumes of a solution containing the diluted kidney homogenate with CO2-saturated water. The decrease in absorbance without the kidney homogenate was determined as blank. The extinction change per second was calculated and normalized to the protein concentration. In initial measurements, a linear dependency between proton release and CAR activity was verified using a commercially available bovine enzyme (2,500 U/mg protein, Sigma). The specificity of the enzyme activity was verified by blocking with acetazolamide (Sigma).

Deiodinase and L-thyroxine determination. The activity of deiodinase (Dio) isozymes was measured as described elsewhere (76). In brief, kidneys or hypothalami of 3-day-old WT and NDI mice were pulverized and homogenized in assay buffer (250 mM glucose, 20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM DTT) by ultrasonification. Following centrifugation at 10,000 g, precipitates were resuspended in assay buffer. Specific Dio activity was determined by measuring the release of iodide from 125I-labeled reverse rT3 in the presence of 1 µM or 1 nM unlabeled rT3 in kidney or brain assays, respectively. To differentiate between Dio1 and Dio2 activity, Dio1 activity was blocked with the specific inhibitor 6-propyl-2-thiouracil (PTU). Dio1 activity was calculated as the difference in Dio activities in the absence and presence of PTU.

Mouse L-thyroxine (3,3',5,5'-tetraiodo-L-thyronine; T4) levels were determined using the neoT4 Kit (PerkinElmer, Rodgau-Jügesheim, Germany). Mouse-tail blood was spotted on filter paper and dried. After automatic punching of blood spots of defined size, the assay was performed according to the manufacturer's protocol.

Determination of urine osmolality. Osmolality of urine samples from adult female animals was determined with a vapor pressure osmometer Vapro 5520 (Wescor) according to the manufacturer's protocol.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Validation of microarray data quality. To identify adaptive gene regulation in response to Avpr2 deficiency, renal and hypothalamic gene transcription was assayed on a whole-transcriptome scale in 3-day-old NDI and their WT littermates. RNA was prepared from the respective tissues, reverse transcribed, and used for Affymetrix microarray hybridization.

To verify the quality of the microarray data, we first analyzed the presence and relative expression levels of selected tissue-specific transcripts. As expected, several AQPs and uromodulin transcripts were preferentially found in the kidney data set (67, 89), while the neurophysin II (AVP) and synaptotagmin (SYT1) genes (1, 31) were found to be preferably expressed in the hypothalamus (see supplemental Table S2).

Next, we analyzed whether genes that are known to be differentially expressed in WT and NDI mice showed a respective pattern in the microarray data. We have recently shown that Avpr2 mRNA levels in the kidneys of Avpr2-deficient mice are lower than in WT mice, probably due to nonsense-mediated mRNA decay (74). The array and qPCR data indeed revealed a ~50% lower expression of Avpr2 mRNA in NDI mice (P < 0.05) (see Table 2). It is well established that dehydration and hypernatremia increase AVP gene expression levels in the hypothalamus (34, 78). In the microarray experiments, AVP expression was significantly increased in NDI mice (1.2-fold in microarray and ~4-fold in qPCR experiments; see Table 2).


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  		Table 2. Expression of genes involved in renal water and electrolyte reabsorption

 
Transcriptome-wide gene expression analysis in kidney and hypothalamus of WT and NDI mice. Of 22,626 probe sets assayed in the microarray hybridization experiments, 12,045 probe sets (corresponding to 8,267 genes) and 11,112 probe sets (corresponding to 7,536 genes) were detected as present (≥2 present calls in either group) in the kidney and hypothalamus, respectively, using the MAS5 algorithm. Of these, 9,232 (6,439 genes) were detected in both tissues.

To assess the extent of differences in gene expression between WT and NDI mice, we subjected the log2-transformed, MAS5-processed microarray data to statistical analysis. In kidney and hypothalamus, 2.6% (216 of 8,267 genes) and 2.3% (172 of 7,536 genes) were significantly different between the groups, respectively, at P ≤ 0.01 (supplemental Tables S3 and S4, respectively). The higher number of regulated genes in the kidney, however, is not significant (P = 0.198, Fisher's exact t-test).

Of the 6,438 genes detected as present in both organs, 1.2% (78 genes) were differentially regulated between WT and NDI mice (P ≤ 0.05) with 0.33% (21 genes) upregulated and 0.62% (40 genes) downregulated in both organs and 0.26% (17 genes) oppositely regulated. The group of similarly regulated genes includes the monocarboxylic acid transporter MCT6 (Slc16a6) and the sodium-coupled neutral amino acid transporter 2 SNAT2 genes. SNAT2 expression is known to be increased under hyperosmotic conditions for rapid restoration of cell volume (8).

In contrast to the low number of genes that are simultaneously regulated in both organs, our data clearly show a high level of tissue-specific gene regulation under the pathophysiological circumstances of NDI. Two hundred and six of the 2,481 genes that were solely detected in the kidney and 101 of 1,592 in the hypothalamus were differentially regulated (P ≤ 0.05).

Initial inspection of the kidney microarray expression data disclosed a number of differentially regulated genes that are directly or indirectly involved in water and sodium transport (Supplemental Table S3). Most of these genes are upregulated in the NDI group. In an explorative approach, we aimed to identify functional systems within the kidney and hypothalamus that show global expression differences between WT and NDI mice.

In the MAS5-processed expression data, 225 probe sets, corresponding to 216 genes, were significantly different between the two groups in kidney and 175 probe sets (172 genes) in hypothalamus (P ≤ 0.01). These genes were subjected to gene ontological analysis (Table 1) using OntoExpress software (25). Interestingly, pathways specifically related to transcellular transport and steroid and prostaglandin biosynthesis were found significantly regulated in the kidney whereas no specific regulation was apparent in the hypothalamus.


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  		Table 1. Gene ontology analysis of genes specifically regulated in NDI

 
In an additional approach, gene set enrichment analysis (61, 84) of the full array data was performed (Supplemental Table S5) using predefined gene sets. The evaluation of genes that are represented in the Collecting Duct Database (50) and that are highly expressed in the kidney showed significant regulation in the groups of transporters and channels, protein kinases, and nuclear receptors. Within the functional pathways defined by the KEGG database (39) and the groups of genes that have a known key function in renal physiology, the corticoid pathway, ATPases, genes involved in the metabolism of prostaglandins and renin, and AQPs are significantly regulated. Testing of additional pathways defined in the MSigDB database (84) pointed to significant involvement of the Wnt, peroxisome proliferator-activated receptors (PPAR), prostaglandin/leukotriene, pentose phosphate, and steroid biosynthesis pathways.

Using the above data, we primarily focused our attention on genes that are known to be expressed in the kidney and that are involved in well-studied mechanisms of electrolyte and water reabsorption. Interestingly, the majority of components involved in the downstream signaling of Avpr2, such as adenylyl cyclases, G proteins, phosphodiesterases, and anchoring proteins, showed no significant differences in the microarray hybridization experiments.

Expression analysis of genes involved in water and electrolyte reabsorption. To verify the differences in gene expression determined by microarray analysis, we performed qPCR for selected transcripts that are involved in water and electrolyte reabsorption (see Table 2). We found significantly enhanced expression levels in NDI mouse kidney for AQP1, AQP2, ouabain-sensitive Na+-K+-ATPase (Atp1a1), sodium phosphate transporter 1 (NPT1), bumetanide-sensitive Na+/K+/Cl– cotransporter (NKCC1), Na+/H+ exchanger 3 (NHE3), sodium bicarbonate cotransporter 7 (NBC3), claudin-15 (Cldn15), CAR2, and prostasin (Prss8) transcripts. Pendrin (Slc26a4) and all ENaC transcripts (Scnn1a, Scnn1b, and Scnn1g) were significantly downregulated.

To evaluate whether these differences are kidney specific, whole brain or hypothalamus RNA was prepared from the same individuals, reverse transcribed, and subjected to qPCR (Table 2). In whole brain, AQP1, NHE3, NPT1, and prostasin were regulated synergistically in the kidney and NKCC1 in the hypothalamus, whereas Cldn15 and AQP2 were oppositely regulated in the kidney. The anion exchanger Slc4a4 was specifically upregulated in whole brain.

Renal reabsorption is subject to complex endocrine regulation. AVP expression was expectedly found strongly upregulated and Avpr1a downregulated in the hypothalamus. We also analyzed key components of the renin-angiotensin system that regulates both renal perfusion and tubular Na+ reabsorption (Table 2). The renin and angiotensin-converting enzyme 1 (ACE1) transcripts were significantly upregulated in the kidney, and angiotensinogen and ACE1 in the hypothalamus, whereas angiotensin II receptor 2 expression was significantly lower in the kidney but stronger in the hypothalamus. In accordance with the strong activation of the renin-angiotensin-aldosterone system (RAAS), the aminopeptidases Anpep and Enpep were markedly upregulated. Similarly, Hsd11b1 and Hsd11b2 were upregulated in the brain and Sgk in both organs, the kidney and brain. The glucocorticoid-induced leucine zipper factor transcript Tsc22d3-1 was significantly upregulated in the kidney.

Functional analysis of selected proteins involved in water and electrolyte transport. Tissue-specific transcript concentrations may not readily reflect functional responses. To assess the functional relevance of the renal gene regulation pattern found in the qPCR experiments, a number of proteins related to renal Na+ reabsorption were quantified on the protein level or functionally assayed.

First, AQP1, which is constitutively integrated into the apical and basolateral plasma membrane of proximal tubular cells, was analyzed in Western blot experiments (Fig. 1A), and band densities were quantified to compare expression levels between WT and NDI mice. Although total AQP1 expression was not different from WT, a marked increase in the nonglycosylated form was detected (Fig. 1B).


Figure 1
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  		Fig. 1. Renal aquaporin-1 (AQP1) expression in wild-type (WT) and nephrogenic diabetes insipidus (NDI) mice. A: kidney tissue homogenates of WT and NDI mice (3-day-old pups) were separated by 15% SDS-PAGE and electroblotted. Two different protein concentrations were used to monitor linearity of immunostaining. AQP1 was detected using an anti-AQP1 antibody (see MATERIALS AND METHODS). The major bands represent the monomeric nonglycosylated (~28 kDa) and glycosylated (~32 kDa) forms of AQP1 (24); faint higher-molecular-weight bands represent most likely multimers of AQP1. B: immunostaining of the 2 majors bands (28 and 32 kDa) from 8 independent Western blots was quantified by densitometry and compared with WT AQP1 expression. Values are means ± SE of the NDI relative to WT samples.

 
CARs, particularly CAR2 and CAR4, are essential components in renal solute transport throughout the nephron (69). In the microarray analysis, renal CAR2, and -3 transcription was increased in NDI mice with significant regulation of CAR2 (P ≤ 0.05). This was also verified by qPCR. CAR activity in homogenized kidneys of WT and NDI pups was then determined using a stopped-flow enzyme kinetic measurement setup (see MATERIALS AND METHODS). In agreement with the observed increase in CAR gene transcription, acetazolamide-sensitive CAR enzyme activity was significantly increased in kidneys of NDI mice compared with their male littermates (Fig. 2A).


Figure 2
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  		Fig. 2. Enhanced renal carbonic anhydrase activity in NDI mice. A: carbonic anhydrase activity was measured in kidneys from male (+/Y, n = 11) and female WT (+/+, n = 15), heterozygous (+/–, n = 15), and NDI (–/Y, n = 11) 3-day-old mice. B: renal carbonic anhydrase activity in adult female WT and heterozygous mice (n = 8). Enzyme activity (means ± SE) is given in arbitrary units (**P < 0.01, ***P < 0.001).

 
The ouabain-sensitive basolateral Na+-K+-ATPase provides the driving force for sodium reuptake throughout the nephron (91). Indeed, we found a significant increase in mRNA expression levels of the Na+-K+-ATPase-{alpha}1 subunit in NDI mice. This finding was confirmed by functional experiments. As shown in Fig. 3A, renal ouabain-sensitive ATPase activity was significantly increased in kidneys of NDI mice compared with the activity in WT kidneys.


Figure 3
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  		Fig. 3. Enhanced Na+-K+-ATPase activity in kidney membranes from WT and NDI mice. Ouabain-sensitive ATPase activity was measured as inorganic phosphate release by ATP hydrolysis in kidney membrane fractions. Kidney membrane preparations from male (+/Y, n = 11) and female WT (+/+, n = 9), NDI (–/Y, n = 9) and female heterozygous (+/–, n = 9) 3-day-old pups (A) or female adult WT (+/+, n = 8) and heterozygous (+/–, n = 8) mice (B) were tested in triplicate. Values are means ± SE (**P < 0.01).

 
NDI pups die in the first postnatal week secondary to malnutrition and malhydration. We used heterozygous female NDI mice as a control group to discriminate between specific effects caused by the NDI pathophysiology and unspecific symptoms of the generally life-threatening situation. Heterozygous female mice display a mild NDI phenotype including polyuria, polydipsia, and production of hypostenuric urine but are otherwise healthy (74, 99). Using qPCR, we quantified in 3-day-old and adult heterozygotes transcripts that were significantly regulated in male NDI pups (Table 3). In heterozygous pups, none of the selected genes, including neurophysin II, was found to be regulated. This was not unexpected because female heterozygote pups present no significant changes in urine osmolality (99). In contrast, heterozygote adult mice are polyuric and hypostenuric (74) and displayed a significant increase in neurophysin II expression. Significant gene regulation with respect to female WT animals was seen for the CAR2, NHE3 (SLC9A3), and NPT1 (SLC17A1) genes (Table 3). CAR activity was significantly increased in adult heterozygous mice (Fig. 2B), and ouabain-sensitive ATPase activity showed a consistent trend in both newborn and adult heterozygous females (Fig. 3). This indicates that key components of tubular sodium and water reabsorption are upregulated not only in NDI pups but also in adult heterozygous mice. Since heterozygote adults present several symptoms of NDI but are not terminally affected, hypostenuric polyuria appears to result, at least in part, from the increased carboanhydrase activity and expression of the Na+-K+-ATPase-{alpha}1 subunit, NHE3, and NPT1.


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  		Table 3. Gene regulation in NDI and female heterozygous mice

 
Regulation of eicosanoid metabolism and signal transduction in NDI mice. Prostanoids derived from renal COX-mediated arachidonic acid metabolism play important roles in the maintenance of glomerular blood flow and tubular salt and water reabsorption (10). Furthermore, prostanoids are not only involved in hypothalamic modulation of pain and body temperature but have also been implicated in the hypothalamic regulation of AVP release in response to various stimuli, including osmotic stress (86). Indeed, gene ontological analysis of the microarray data revealed significant differences between WT and NDI mice in the renal prostaglandin (PG) biosynthesis group (Table 1), which were mainly due to significant upregulation of PGD2 synthase and EP4 receptor transcription in NDI mice (Table 4).


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  		Table 4. Expression of genes involved in eicosanoid synthesis and metabolism

 
To verify the results of the initial microarray analyses, qPCR experiments were performed for 27 key components of the eicosanoid metabolism and signal transduction pathways. In both NDI mouse kidney and hypothalamus, significant upregulation of COX2 (Ptgs2), accompanied by downregulation of COX1 (Ptgs1) was found (Table 4). In NDI mouse kidneys, significant upregulation of PGD2 synthase (Ptgds) was confirmed, while leukotriene C4 synthase (Ltc4s) was downregulated. In the hypothalamus of NDI mice, the prostanoid-degrading enzymes dipeptidase 1 (Dpep1) and {gamma}-glutamyltransferase (GGT1) were strongly up- and thromboxane A2 synthase 1 (Tbxas1) and epoxide hydrolase 2 (Ephx2) down-regulated. A number of hypothalamic eicosanoid receptors were differentially regulated between WT and NDI mice, including the leukotriene B4 receptor 1 (Ltb4r1) and the PGD receptor (Ptgdr, Table 4).

These data are indicative of an increased activity of the renal PG system (COX2/COX1 ratio, PGD synthase) in NDI mice. In contrast to the kidney, qPCR experiments revealed no significant changes in PGD synthase expression but increased expression of Cys-leukotriene converting enzymes in the hypothalami of NDI mice.

Functional upregulation of the thyroid hormone system in NDI mice. The initial microarray expression data indicated that several components known to be induced by thyroid hormones (13, 59) were upregulated in the kidney of NDI mice, including Dio1 and thyroid hormone responsive spot 14 (Thrsp). qPCR experiments confirmed three- to fourfold increased expression of Dio1 and nearly twofold upregulation of Thrsp transcripts in both the kidney and hypothalamus of NDI mice (Table 5). Additionally, Dio2 was upregulated in both organs and Dio3 expression was stronger in kidneys of NDI mice (see Supplementary Table S7).


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  		Table 5. Expression of genes involved in thyroid hormone metabolism

 
To analyze whether increased Dio1 and Dio2 transcript concentrations led to increased Dio function, Dio activity was determined in the kidney and whole brain of WT and NDI 3-day-old mice. As shown in Fig. 4A, renal Dio1 activity was increased about twofold in NDI mice.


Figure 4
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  		Fig. 4. AVP receptor (AVPR2) deficiency leads to increased thyroid hormone metabolism. A: renal deiodinase-1 (Dio1) activity was determined in kidney homogenates from 3-day-old WT and NDI mice (n = 8 in both groups). B: T4 levels were measured in mouse tail blood using a commercial test kit. Values of 3-day-old WT (n = 54) and NDI (n = 15) mice are given as means ± SE (**P < 0.01).

 
As Thrsp and Dio expression is induced by thyroid hormones, plasma T4 levels were measured in WT and NDI animals. We found a highly significant increase in T4 levels in NDI mice (~1.5-fold) relative to WT mice (Fig. 4B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Some diuretics, particularly thiazide- and potassium-sparing diuretics, have beneficial effects in patients with inherited forms of NDI. The mechanisms underlying their therapeutic actions are not well understood on the basis of the current pathophysiological concept of NDI. In a neuroendocrine circuit, hypothalamic neurons sense plasma osmolality and trigger the release of AVP, which regulates water absorption in the renal collecting duct. In the case of malfunction of the AVP endocrine system, as in X-linked NDI, it is likely that both the sensor and the hormone target organs respond to counteract the resulting water and electrolyte imbalance. These adaptive changes may either improve the pathophysiological situation or, on the other hand, contribute to the clinical features of NDI and thus form potential therapeutic targets.

In this study, we aimed to identify renal and hypothalamic compensatory mechanisms in NDI. We used a microarray-based approach for transcriptome-wide screening of renal and hypothalamic gene expression. Although components of a variety of functional systems including metabolism, protein synthesis and cell division were found to be differentially regulated, we mainly focused on systems with potential functional relevance for NDI therapy.

Nephron function is highly site specific, and each nephron segment has a characteristic expression profile for its functional components. However, owing to the small size of the organs of the 3-day-old mice investigated in this study, it was technically not feasible to investigate isolated parts of the nephron. Therefore, at this stage, we provide and discuss expression data of whole kidneys. However, further studies using adult mice bearing regional-specific genetic AVPR2 inactivation may help to finely localize the effects presented here.

Renal sodium reabsorption is increased in NDI. Our mRNA expression analyses revealed a number of differentially expressed genes that are involved in renal sodium reabsorption. In the proximal tubule, CARs are key players in sodium bicarbonate reabsorption and acid secretion. The electrochemical energy provided by the basolateral Na+-K+-ATPase is used by a Na+/H+ antiporter (NHE3; Slc9A3) in the apical membrane to transport H+ into the tubular lumen in exchange for Na+. The major mechanism for the transport of HCO3– across the basolateral membrane is via electrogenic Na+:3 HCO3– cotransporters (NBC) (79), including Slc4A4 (NBC1) and Slc4A7 (NBCn1). In addition, the apical sodium phosphate transporter NPT1 (Slc17A1) (60, 80) and claudins-2 and -15, which form paracellular charge-selective sodium chloride pores (20, 88) in the proximal tubule (27) and the vasa recta of the medulla (35), are involved in Na+ reabsorption. All of these components were found upregulated in the kidneys of NDI mice (Table 2). Increased CAR and ouabain-sensitive Na+-K+-ATPase activities were also verified by enzyme activity measurements (Figs. 2 and 3). In sum, these data point to increased proximal Na+ reabsorption that is supposedly linked to increased proximal tubular water reabsorption. In agreement with this finding, renal AQP1 was upregulated in NDI mice at the transcript level and an increase in the nonglycosylated form was seen in Western blot experiments that points to enhanced de novo synthesis of AQP1 protein (Table 2 and Fig. 1). When purified and reconstituted, there is no functional difference between the glycosylated and nonglycosylated AQP1 form (87). It remains to be determined, however, whether sodium/water reabsorption is still isotonic in NDI.

In the loop of Henle, sodium, potassium, and chloride ions are reabsorbed by active transport through the bumetanide-sensitive Na+/K+/2 Cl– cotransporters (NKCC1; Slc12a1, Slc12a2). Microarray and qPCR analyses revealed significant increases in renal expression of the Slc12a2 transcript in NDI mice. We have previously analyzed renal BSC1 and AQP2 protein expression by Western blotting but found no significant differences between WT and NDI pups (99). Furthermore, the expression difference of ~20% measured by qPCR experiments may escape detection in Western blot analysis. Increased BSC1 expression, however, increases renal medullary tonicity and thus might contribute to increased water reabsorption through collecting duct cells if AVP-dependent AQP2 translocation were intact. Since this is not the case in X-linked NDI, increased sodium reuptake in the loop of Henle may also contribute to hypernatremia and urine hyposmolality.

Previous studies have reported that, in addition to AQP2 translocation, chronic AVP treatment stimulates amiloride-sensitive Na+ transport in isolated renal cortical collecting duct cells and increases the abundance of β- and {gamma}-subunits of ENaC in the kidney (75). Although increased AVP serum concentration is an important diagnostic feature of X-linked NDI, and qPCR experiments showed an approximately fourfold higher hypothalamic expression level of the AVP precursor neurophysin II in NDI mice (Table 4), our microarray hybridization and qPCR data indicate reduced expression levels of all three ENaC subunits (Scnn1a/b/g; Table 2). However, expression of prostasin, a protease that activates ENaC (2, 23), was significantly increased (Table 2). Furthermore, the glucocorticoid-induced leucine zipper protein (GILZ or TSC22D3) and glucocorticoid-induced kinase (SGK1) transcripts displayed increased expression levels in the mouse NDI model (Table 2). There is evidence that aldosterone mediates its sodium reabsorption effect on ENaC via regulation of GILZ and Sgk expression (9). GILZ stimulates ENaC-mediated Na+ transport by inhibiting extracellular signal-regulated kinase (ERK) signaling (81). However, it remains open whether the functional net effect is a change in ENaC-mediated Na+ reabsorption in NDI mice.

Consistent with increased HCO3– reabsorption in the proximal tubule, the apical Cl–/HCO3– exchanger pendrin, which mediates HCO3– secretion and Cl– absorption in the connecting tubule and the cortical collecting duct (72, 96), is significantly downregulated in NDI mice (see Table 2). The expression of the Na+-Cl– cotransporter (Slc12A3), the major target of thiazide diuretics, showed no significant changes.

Taken together, our expression and functional data clearly indicate an increase in proximal tubular HCO3– and Na+ reabsorption and H+ secretion in NDI mice. Although presumably intended to enhance Na+-coupled water reabsorption and to prevent systemic metabolic acidosis, this active compensatory mechanism is likely to deteriorate the electrolyte imbalance and systemic pathology in NDI.

Eicosanoid metabolism is differentially regulated in kidney and hypothalamus of NDI mice. In the kidney, PGs play a crucial role in the regulation of renal hemodynamics, renin release, and tubular sodium and water reabsorption (33). Besides its particular function in the macula densa, PGE2 plays an important role in attenuating the antidiuretic action of AVP in the renal collecting duct. A positive correlation between PGE2 excretion and urinary flow rate has been observed (95), and COX inhibitors are successfully used to reduce urine volume in NDI patients. The present study revealed a significant downregulation of COX1, the constitutively expressed COX enzyme, in kidneys of NDI mice, whereas COX2 expression was significantly increased. This is consistent with previous findings that dehydration and increased interstitial tonicity induce expression of COX2 (46, 98). The most prominent increase in mRNA expression (~3-fold), however, was found for PGD2 synthase. It has been previously shown in dogs that PGD2 administration results in a significant dose-dependent increase in renal artery flow, urine output, creatinine clearance, plasma renin activity, and Na+ and K+ excretion (70). It is therefore possible that PGD2, in addition to PGE2, significantly contributes to the increased urine output in NDI.

In the hypothalamus,TRPV1 and TRPV4 appear to function as osmosensors (53, 77, 83), or part of an osmosensor complex (19) that controls AVP release from the posterior lobe of the pituitary gland. In TRPV4-deficient mice, plasma AVP levels were significantly lower than in their WT littermates after hyperosmotic challenge (54). Consistent with this observation, we found that hypothalamic TRPV4 expression was increased in NDI mice (Table 2). It is, however, still unsolved how the signal is propagated from the hypothalamic osmosensors to the AVP-producing magnocellular neurons in the supraoptic and the paraventricular nuclei. There is strong evidence that TRPV4 does not account alone for osmosensitivity. Lack of TRPV1 also results in serum hyperosmolality under ad libitum conditions (77). Although AVP-releasing neurons in the supraoptic nucleus are directly osmosensitive, a process that involves stretch-inhibited cation channels (65), TRPV4 activity seems to be also mediated indirectly by chemical signals (93). There is evidence that PGD2 and PGE2 are involved in the regulation of AVP secretion (11, 43). Furthermore, it was shown that TRPV4 function is modulated by eicosanoids, including epoxyeicosatrienoic acid derivatives (EETs) (92). Given the differentially regulated COX isoenzymes, the downregulated expression of epoxide hydrolase 2, and the increased cysteinyl leukotriene metabolism in hypothalamus of NDI, our data support a significant involvement of eicosanoids in the hypothalamic response to hypernatremia and dehydration.

Thyroid hormone metabolism is increased in NDI mice. In Avpr2-deficient mice, we found a significant increase in the serum concentration of the thyroid hormone T4 (Fig. 4B), accompanied by enhanced thyroid hormone-induced transcriptional activity and renal thyroid hormone metabolism. The activation of the primary secretory product of the thyroid gland, T4, into the biologically active form, T3, is catalyzed by two deiodases, Dio1 and Dio2, whereas inactivation of both T4 and T3 occurs via Dio3 and, to some extent, by Dio1. Dio enzymes thus modulate thyroid hormone signal transduction on the prereceptor level. Dio1 transcripts were found to be significantly upregulated in NDI mice in both the kidney and hypothalamus (Table 5), and a twofold increase in renal Dio1 activity was also confirmed by enzyme activity measurements (Fig. 4A).

The thyroid responsive protein spot 14 (Thrsp) represents an early marker of thyroid hormone action that rapidly increases following T3 application (62). In the liver, Thrsp leads to the induction or activation of key lipogenic, glycolytic, and gluconeogenetic enzymes (13). In agreement with the enhanced thyroid hormone turnover, THRSP gene expression was found upregulated in both the kidney and hypothalamus of NDI mice. Thyroid hormones have a well-defined action in renal function. Rats treated with T4 respond with a significant increase in renal renin and ACE expression and activity (16, 45). There is also evidence that T3 directly stimulates renal NaCl and NaHCO3 reabsorption by increasing the expression and activity of Na+-K+-ATPase (32, 51, 52, 55) and NHE3 (15). It has been shown that the highest density of thyroid hormone receptors is in the proximal tubule followed by the collecting duct, where receptor activation positively regulates Na+-K+-ATPase activity (5). This probably is also the site of highest transcript levels of Dio1 in rat kidney (49). Furthermore, aldosterone-induced Na+-K+-ATPase activity in the distal tubule is markedly diminished after thyroidectomy (6). These and other evidence (4, 18, 44) point to direct endocrine cross talk between thyroid hormone signaling and the activity of the RAAS. In this context, enhanced activity of the thyroid hormone system may represent an endocrine mechanism that contributes to increased renal Na+ reabsorption via direct (increased Na+-K+-ATPase gene and NHE3 transcription) and indirect (RAAS activation) mechanisms in NDI mice.

Proposed mechanism of NDI drug action and implications for therapy. In principle, dehydration in NDI can be compensated by a decrease in the glomerular filtration rate (GFR) or by enhanced tubular water transport either secondary to Na+ resorption or increased renal medullary osmolarity. GFR autoregulation through tubuloglomerular feedback, however, is impaired in NDI due to urine hypotonicity and hypernatremia, which itself stimulates renal PGE2 release, resulting in a detrimental GFR increase. Our findings rather suggest that active Na+ reabsorption in the proximal tubular nephron is significantly stimulated in NDI to counteract systemic dehydration (Fig. 5). Besides the increased expression levels of specific components of proximal tubular and loop of Henle Na+ transport, there is evidence that distal tubular Na+ reabsorption is also modulated in NDI. Prostasin, GILZ and Sgk, all positive regulators of ENaC, are upregulated in NDI mice.


Figure 5
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  		Fig. 5. Pathophysiological changes in NDI mice. Based on the expression and functional data, the following NDI pathomechanism is proposed: Water loss, due to the lack of vasopressin-mediated AQP2 insertion into the apical membrane of collecting duct cells, induces increased sodium reabsorption mainly in the proximal tubule. This regulative process is mediated by an increased activity of the renin-angiotensin-aldosterone system (RAAS) and thyroid hormone systems. Both increase the expression and activity of key components such as carbonic anhydrases (CAR), ouabain-sensitive Na+-K+-ATPase, AQP1, and Na+/H+ exchanger type 3 (NHE3), which are all involved in tubular sodium and water reuptake. Hypernatremia in NDI is therefore the result of increased sodium reabsorption in the proximal tubule and net loss of water due to the continuous dysfunction of water reabsorption in the collecting duct. This vicious circle is further driven by enhanced formation of prostaglandins (PGE2, PGI2) and angiotensin II that amplify glomerular filtration rate. NKCC1, bumetanide-sensitive Na+/K+/Cl– cotransporter; ENaC, epithelial sodium channel; COX, cyclooxygenase.

 
Owing to the lack of Avpr2, increased proximal Na+ retention is not accompanied by adequate water reabsorption in the collecting duct, and systemic hypernatremia results. We speculate that blockade of this vicious circle may contribute to the therapeutic effect of diuretics in diabetes insipidus. Most thiazide diuretics have additional CAR-inhibiting properties (68) that may enhance their therapeutic usefulness. Although acetazolamide, a prototypical CAR inhibitor, has been found less potent than thiazide diuretics in treating polyuria (21), the potential therapeutic action of CAR inhibition in NDI patients needs to be evaluated in greater detail.

Multiple findings directly and indirectly point to strong activation of the RAAS in NDI mice. Both renin and angiotensin I-converting enzyme 1 transcripts were found up- and the angiotensin II receptor type 2 downregulated (Table 2). Several genes that are upregulated in the kidneys of NDI mice are known to be induced by angiotensin II (NHE3, NBC) (30) and aldosterone (ROMK, GILZ, PRSS, SGK, Na+-K+-ATPase genes) (3, 7, 9, 63, 90). In addition to increased aldosterone secretion, the tissue-specific activation of corticoids appears to be significantly enhanced in NDI mice. 11-β-Hydroxysteroid dehydrogenases (11βHSD1 and 11βHSD2) catalyze the conversion of inactive glucocorticoids (e.g., cortisone) to their active forms (e.g., cortisol) and vice versa. In the kidney, 11βHSD1 predominantly generates biologically active corticosterone whereas 11βHSD2 metabolizes cortisol or corticosterone (the corticosteroid in rodents) into inactive metabolites that are unable to bind to mineralocorticoid or glucocorticoid receptors (26). Interestingly, we specifically observed an increase in renal expression of 11βHSD1 in NDI mice (Table 2), a finding that acts synergistically with the increased RAAS activity and further enhances expression of mineralocorticoid-regulated genes. This suggests that NDI patients might benefit from inhibition of the RAAS by ACE or aldosterone receptor inhibitors. This concept is supported by a recent study showing that aldosterone increases urine production and decreases apical AQP2 expression in rats with lithium-induced NDI, whereas spironolactone, an aldosterone antagonist, reversed this effect (64). On the basis of these results and a number of case reports in NDI patients (17, 22), the therapeutic usefulness of RAAS inhibition in NDI should be reevaluated.

The mechanism underlying the therapeutic action of COX inhibitors in NDI appears to be reduction of renal medullary blood flow secondary to diminished renal PG synthesis. Indeed, unspecific COX inhibitors have proved clinically successful. The specific upregulation of COX2 expression that is found in NDI mice, however, suggests that selective COX2 inhibition may be clinically advantageous, as treatment of NDI patients with indomethacin is often associated with gastric side effects. A number of COX2-specific inhibitors have been developed to reduce these undesired effects and appear to be a rational choice in NDI. Rofecoxib and, more recently, celecoxib in combination with HCTZ were already successfully used to treat congenital NDI (66, 82).

Although several of the renal tubular proteins discussed above (e.g., NBC or NBCn1) undergo similar regulation in congenital (this study) and lithium-induced forms of NDI (41), there are significant differences in gene expression between X-linked and lithium-induced NDI (41, 47, 48, 57). In contrast to X-linked NDI, no changes or even a reduction in expression was observed for basolateral Na+-K+-ATPase (47, 48), AQP1 (47), AQP2 (48, 57), NPT1 (47), and pendrin (41) in lithium-induced NDI (48). However, one should reflect that in the latter, the altered Na+ and water reabsorption is drug induced in otherwise healthy adult rats that may more effectively adapt to NDI pathophysiology than newborn mice. As both Avpr2- and AQP2-deficient mice die during the first week after birth, the full NDI phenotype could not be studied in adult mice so far. Recently, an inducible mouse model of AQP2 deficiency was generated and characterized (97). It should be of interest to evaluate changes in gene expression levels in these adult mutant mice.

In sum, transcriptome-wide analysis of renal gene expression and functional analysis of selected key proteins identified enhanced active proximal tubular Na+ reabsorption as an important component in the pathomechanism of NDI. Our data support the rationales of current therapeutic approaches, implicate a therapeutic benefit of inhibition of carbonic anhydrases, RAAS, and specific components of PG synthesis and highlight a previously unappreciated role of renal thyroid hormone action.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was sponsored by Deutsche Forschungsgemeinschaft (Scho 624/2-1, GRK 1097), Bundesministerium für Bildung und Forschung, Interdisziplinäres Zentrum für Klinische Forschung (IZKF) Leipzig and the Leipzig Formel 1 program to K. Sangkuhl and H. Römpler.


   ACKNOWLEDGMENTS
 
We are very grateful to Dr. Mark Knepper for many suggestions and help in interpretation of the data.

Present addresses: H. Römpler, Dept. of Organismic and Evolutionary Biology and the Museum of Comparative Zoology, Harvard University, Cambridge, MA; and K. Sangkuhl, Div. of Reproductive Biology, Dept. of Obstetrics and Gynecology, Medical Center, Stanford University, Stanford, CA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Schöneberg, Institute of Biochemistry, Molecular Biochemistry, Medical Faculty, Univ. of Leipzig, Johannisallee 30, 04103 Leipzig, Germany (e-mail: schoberg{at}medizin.uni-leipzig.de)

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.

* N. Schliebe and R. Strotmann contributed equally to this work. Back


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