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Lithium salts, used for treating bipolar disorder, frequently induce nephrogenic diabetes insipidus (NDI) thereby limiting therapeutic success. NDI is associated with loss of expression of the gene coding for the molecular water channel, aquaporin-2, in the renal collecting duct (CD). Here, we use systems biology methods in a well-established rat model of lithium-induced NDI to identify signaling pathways activated at the onset of polyuria. Using single-tubule RNA-Seq, full transcriptomes were determined in microdissected cortical collecting ducts of rats after 72 hours without or with initiation of lithium chloride administration. Transcriptome-wide changes in mRNA abundances were mapped to gene sets associated with curated canonical signaling pathways, showing evidence for activation of NF-ºB signaling with induction of genes coding for multiple chemokines and most components of the Major Histocompatibility Complex Class I antigen-presenting complex. Administration of anti-inflammatory doses of dexamethasone to lithium chloride -treated rats countered the loss of aquaporin-2. RNA-Seq also confirmed prior evidence of a shift from quiescence into the cell cycle with arrest. Time course studies demonstrated an early (12 hour) increase in multiple immediate early response genes including several transcription factors. Protein mass spectrometry in microdissected cortical collecting ducts provided corroborative evidence and identified decreased abundance of several antioxidant proteins. Thus, in the context of prior observations, our study can be best explained by a model in which lithium increases ERK activation leading to induction of NF-κB signaling and an inflammatory-like response that represses Aqp2 transcription.

X-linked nephrogenic diabetes insipidus (X-NDI) is a disease caused by inactivating mutations of the vasopressin (AVP) type 2 receptor ( V2R ) gene. Loss of V2R function prevents plasma membrane expression of the AQP2 water channel in the kidney collecting duct cells and impairs the kidney concentration ability. In an attempt to develop strategies to bypass V2R signaling in X-NDI, we evaluated the effects of secretin and fluvastatin, either alone or in combination, on kidney function in a mouse model of X-NDI. The secretin receptor was found to be functionally expressed in the kidney collecting duct cells. Based on this, X-NDI mice were infused with secretin for 14 days but urinary parameters were not altered by the infusion. Interestingly, secretin significantly increased AQP2 levels in the collecting duct but the protein primarily accumulated in the cytosol. Since we previously reported that fluvastatin treatment increased AQP2 plasma membrane expression in wild-type mice, secretin-infused X-NDI mice received a single injection of fluvastatin. Interestingly, urine production by X-NDI mice treated with secretin plus fluvastatin was reduced by nearly 90% and the urine osmolality was doubled. Immunostaining showed that secretin increased intracellular stores of AQP2 and the addition of fluvastatin promoted AQP2 trafficking to the plasma membrane. Taken together, these findings open new perspectives for the pharmacological treatment of X-NDI.

The first non-peptide vasopressin receptor antagonist (VRA) was recently approved by the United States Food and Drug Administration, and several others are now in late stages of clinical development. Phase 3 trials indicate that these agents predictably reduce urine osmolality, increase electrolyte-free water excretion, and raise serum sodium concentration. They are likely to become a mainstay of treatment of euvolemic and hypervolemic hyponatremia. Although tachyphylaxis to the hydro-osmotic effect of these agents does not appear to occur, their use is accompanied by an increase in thirst, and they do not always eliminate altogether the need for water restriction during treatment of hyponatremia. Experience with use of these agents for treatment of acute, severe, life-threatening hyponatremia as well as chronic hyponatremia is limited. Further studies are needed to determine how they are best used in these situations, but the risk of overly rapid correction of hyponatremia seems low. Results of long-term trials to determine the ability of VRAs to reduce morbidity or mortality in congestive heart failure or to slow the progression of polycystic kidney disease are awaited with great interest.

Down-regulation of urea transporters in the renal inner medulla of lithium-fed rats. Background Lithium is commonly used to treat bipolar psychiatric disorders but can cause reduced urine concentrating ability. Methods To test whether lithium alters UT-A1 or UT-B urea transporter protein abundance or UT-A1 phosphorylation, rats were fed a standard diet supplemented with LiCl for 10 or 25 days, and then compared to pair-fed control rats. To investigate another potential mechanism for decreased urea transport, inner medullary collecting duct (IMCD) suspensions from lithium-fed or control rats were incubated with 32 P-orthophosphate to measure the phosphorylation of UT-A1. Results In lithium-fed rats (25 days), UT-A1 abundance was reduced to 50% of control rats in IM tip and to 25% in IM base, and UT-B abundance was reduced to 40% in IM base. Aquaporin-2 (AQP2) protein abundance was reduced in both IM regions. Vasopressin (100 pmol/L) increased UT-A1 phosphorylation in IMCD suspensions from control but not from lithium-fed rats; a higher vasopressin concentration (100 nmol/L) increased UT-A1 phosphorylation in control and lithium-fed rats. Conclusions Decreases in UT-A1, UT-B, and AQP2 protein abundance, and/or vasopressin-stimulated phosphorylation of UT-A1, can contribute to the reduced urine concentrating ability that occurs in lithium-treated rats.

A 17-year-old boy was admitted with chronic kidney disease (CKD), polyuria, and polycytemia. Prenatal ultrasound (US) history included enlarged hyperechogenic kidneys and polyhydramnios. Since birth, he suffered from polyuria and polydipsia. At the age of 3 years, renal function was normal; US showed normal-sized cystic kidneys. Genetic testing for nephronophthisis excluded NPHP1 gene deletions. At the age of 15 years, he developed hypertension and polycythemia requiring phlebotomies; serum creatinine was 1.5 mg/dl; US revealed normal-sized cystic kidneys. UMOD gene mutations were not found. At admission, serum creatinine was 1.5 mg/dl; creatinine clearance 67 ml/min; plasma sodium and potassium 142 and 4.5 mEq/l, respectively; uric acid 5.1 mg/dl. Urinalysis was negative; daily urine volume 4 liters; urinary osmolality 330 mOsmol/kg. Magnetic resonance imaging revealed normal-sized kidneys and numerous bilateral cysts (Figure 1). No alteration in glucose metabolism was found. Water restriction and desmopressin administration showed a submaximal rise in urine osmolality (481 and 497 mOsm/kg, respectively), suggesting partial nephrogenic diabetes insipidus (NDI). Sequencing of AVPR2 and AQP2 genes did not reveal mutations. Hemoglobin was 18 g/dl. Serum erythropoietin concentrations were elevated (21 mU/ml; n.v.

Angiotensin II plays an important role in the regulation of blood pressure, body salt and fluid balance, and urine concentration. Mice with deletion of the AT1a receptor develop polyuria and urine concentration defects. We studied the mechanisms of these urine concentration defects by treating wild-type and AT1a-knockout mice with arginine vasopressin (AVP) for 2 weeks, controlling their water intake, or giving them an osmotic diuretic (sucrose) in order to determine whether central or nephrogenic mechanisms were involved. Under basal conditions, AT1a-knockout mice were hypotensive, had lower plasma AVP, and excreted more urine with a markedly reduced osmolality compared with wild-type mice. However, basal glomerular filtration rates were similar in both strains of mice. We isolated total lysate and membrane proteins from the inner medulla of wild-type and mutant mouse kidneys, and found that the amounts of aquaporin 2 (AQP2), adenylyl cyclases III and V/VI, and phosphorylated MAP kinases ERK 1/2 proteins were all reduced in the inner medulla of the knockout mice. Infusion of AVP raised plasma levels and blood pressure proportionally in both strains, but polyuria persisted and urine osmolality remained significantly lower in the knockout mice. Although AVP increased urine osmolality slightly in water-deprived knockout mice, this was well below the basal osmolality of wild-type mice. The diuretic response to the hyperosmotic sucrose was also impaired in the knockout mice. Neither AVP nor water rationing restored the levels of the inner medullary signaling proteins and membrane AQP2 proteins in the knockout mice. We suggest that AT1a receptor deletion causes polyuria and urine concentration defects by decreasing basal AVP release and impairing AVP-induced receptor signaling in the inner medulla.

Polarized expression of the vasopressin V2 receptor in Madin-Darby canine kidney cells. Background The vasopressin V2 receptor is expressed in the polarized principal cell of the renal collecting duct. Inactivating mutations of the vasopressin V2 receptor gene cause X-linked nephrogenic diabetes insipidus (NDI). Most of the mutant V2 receptors show transport defects, as analyzed in non-polarized cells, but data pertaining to polarized cells have not previously been presented. Methods Madin-Darby canine kidney cell (MDCK) II clones stably expressing c-myc–tagged human V2 receptors were characterized for [ 3 H]-arginine vasopressin (AVP)-binding and AVP-sensitive adenylyl cyclase activity. The V2 receptors were immunocytochemically localized using the tyramide signal amplification technique in conjunction with an anti–c-myc antibody. Results The introduction of the c-myc epitope at the N- or C-terminus did not affect the functional properties of the V2 receptor expressed in MDCK II clones. However, the use of standard immunofluorescence methodology for these MDCK II clones yielded only weak signals. With the tyramide signal amplification technique, strong signals were obtained, showing the V2 receptor to be mainly localized within the lateral and, to a minor extent, apical membrane. In MDCK II clones stably expressing the c-myc–tagged V2 receptor NDI mutant L44P, fluorescent signals were found exclusively within the cell. Conclusion The wild-type V2 receptor is expressed mainly in the lateral membrane, whereas the L44P mutant is completely retained within the cell. In conjunction with tyramide signal amplification, MDCK II cells constitute a suitable model for the analysis of transport-defective mutants of the V2 receptor.

Cyclic-3′,5′-nucleotide phosphodiesterase isozymes in cell biology and pathophysiology of the kidney. Investigations of recent years revealed that isozymes of cyclic-3′,5′-nucleotide phosphodiesterase (PDE) are a critically important component of the cyclic-3′,5′-adenosine monophosphate (cAMP) protein kinase A (PKA) signaling pathway. The superfamily of cyclic-3′,5′-phosphodiesterase (PDE) isozymes consists of at least nine gene families (types): PDE1 to PDE9. Some PDE families are very diverse and consist of several subtypes and numerous PDE isoform-splice variants. PDE isozymes differ in molecular structure, catalytic properties, intracellular regulation and location, and sensitivity to selective inhibitors, as well as differential expression in various cell types. A number of type-specific "second-generation" PDE inhibitors have been developed. Current evidence indicates that PDE isozymes play a role in several pathobiologic processes in kidney cells. In rat mesangial cells, PDE3 and PDE4 compartmentalize cAMP signaling to the PDE3-linked cAMP-PKA pathway that modulates mitogenesis and PDE4-linked cAMP-PKA pathway that modulates generation of reactive oxygen species. Administration of selective PDE isozyme inhibitors in vivo suppresses proteinuria and pathologic changes in experimental anti-Thy-1.1 mesangial proliferative glomerulonephritis in rats. Increased activity of PDE5 (and perhaps also PDE9) in glomeruli and in cells of collecting ducts in sodium-retaining states, such as nephrotic syndrome, accounts for renal resistance to atriopeptin; diminished ability to excrete sodium can be corrected by administration of the selective PDE5 inhibitor zaprinast. Anomalously high PDE4 activity in collecting ducts is a basis of unresponsiveness to vasopressin in mice with hereditary nephrogenic diabetes insipidus. Apparently, PDE isozymes apparently also play an important role in the pathogenesis of acute renal failure of different origins. Administration of PDE isozyme–selective inhibitors suppresses some components of immune responses to allograft transplant and improves preservation and survival of transplanted organ. PDE isozymes are a target for action of numerous novel selective PDE inhibitors, which are key components in the design of novel "signal transduction" pharmacotherapies of kidney diseases.

MicroRNAs (miRNAs) are endogenous short (20–22 nucleotides) non-coding RNA molecules that mediate gene expression. This is an important regulatory mechanism to modulate fundamental cellular processes such as differentiation, proliferation, death, metabolism, and pathophysiology of many diseases. The miRNA expression profile of the kidney differs greatly from that of other organs, as well as between the different regions in the kidney. In kidneys, miRNAs are indispensable for development and homeostasis. In this review, we explore the involvement of miRNAs in the regulation of blood pressure, hormone, water, and ion balance pertaining to kidney homeostasis. We also highlight their importance in renal pathophysiology, such as in polycystic disease, diabetic nephropathy, nephrogenic diabetes insipidus, hypertension, renal cancer, and kidney fibrosis (epithelial–mesenchymal transition). In addition, we highlight the need for further investigations on miRNA-based studies in the development of diagnostic, prognostic, and therapeutic tools for renal diseases.

Inheritance of mutations in the V2 receptor gene in thirteen families with nephrogenic diabetes insipidus. Congenital nephrogenic diabetes insipidus (NDI) is an X-linked recessive disease characterized by insensitivity of the distal nephron to the antidiuretic effect of arginine vasopressin. The hypothesis that the defect underlying NDI might be a dysfunctional renal vasopressin V2 receptor has recently been proven by the identification of mutations in the V2 receptor gene in NDI patients. We examined thirteen unrelated Dutch NDI families and identified thirteen distinct and unique mutations. These included nine missense mutations, two nonsense mutations and two small deletions and were found in the extracellular domains II, III and IV, the intracellular domains II and IV and in the transmembrane loops I, II, IV and V of the vasopressin type 2 receptor. In the families with multiple NDI patients the mutated gene cosegregated with the disease. Our data suggest a higher mutation frequency in male than in female gametes. No discrepancies between carrier detection by means of DNA analysis with closely linked polymorphic markers and the definite diagnosis based on sequencing data were found.

ADH resistance of LLC-PK 1 cells caused by overexpression of cAMP-phosphodiesterase type-IV. The studies of animal models of nephrogenic diabetes insipidus (NDI) suggest that abnormally high activity of cAMP phosphodiesterase (cAMP-PDE) may cause unresponsiveness to the diuretic effect of AVP. We explored whether overexpression of one of the cAMP-PDE type isozymes, PDE-IV, in [8-Arg]-vasopressin (AVP) sensitive renal epithelial LLC-PK 1 cells can prevent the hormone-elicited cAMP increase. LLC-PK 1 cells were stably transfected with ratPDE3.1 cDNA (which encodes for rolipram-sensitive PDE-IV), inserted in plasmid pCMV5 and then were compared with sham-transfected LLC-PK 1 cells and wild LLC-PK 1 cells. In the stably transfected clone (LLC-PK 1 S#16), the rolipram-sensitive PDE-IV activity was about five times higher than in controls, whereas activities of other types of PDEs were not different. The presence of cognate mRNA for PDE-IV was confirmed by Northern blot. Whereas in the control cells (wild LLC-PK 1 cells and sham-transfected LLC-PK 1 cells), the incubation with 10 -7 M AVP increased cAMP more than tenfold, the LLC-PK 1 S#16 cells with overexpressed cAMP-PDE were resistant to cAMP-increasing effects of AVP and forskolin. However, in the same LLC-PK 1 -S#16 cells the cGMP increases in response to nitroprusside were not diminished. The AVP-dependent cAMP accumulation in LLC-PK 1 S#16 cells with overexpressed PDE-IV was restored by addition of roliprams which decreased cAMP-PDE activity to the levels similar to those in wild LLC-PK 1 cells and sham-transfected LLC-PK 1 -#A1 cells. In contrast, inhibitors of other PDE isozymes (PDE-I or PDE-III) had little or no effect. Our findings show that excessive activity of cAMP-PDE, in this case of isozyme PDE-IV, can cause resistance to AVP which is analogous to that observed in collecting ducts of mice with hereditary nephrogenic diabetes insipidus.

Mechanism of lithium-induced polyuria in the rat. While many studies have demonstrated a nephrogenic diabetes insipidus syndrome (NDI) with prolonged lithium (Li) treatment, experiments in the isolated rat papillary collecting duct have suggested that the defect may be due to a circulating factor that inhibits the action of arginine vasopressin (AVP). Since Li-treatment can produce a form of hyperparathyroidism and parathyroid hormone (PTH) can act as a partial agonist to AVP, in vivo and in vitro studies were performed on rats made polyuric by daily intraperitoneal (i.p.) Li (4 mmol/kg) treatment. Li-treatment for three weeks produced an increase in PTH (194 ± 20 compared with 118 ± 18 pg/ml in control rats; P < 0.01) as well as an increase in the plasma calcium concentration (2.38 ± 0.05 compared with 2.25 ± 0.04 mmol/liter; P < 0.05). Clearance studies were performed on water loaded Li-treated and control rats, and the defect in urine concentration was only observed with a low physiological concentration of AVP (10 mU/kg body wt over 5 min). Maximal urine osmolality was 328 ± 31 compared with 613 ± 81 mOsm/kg (P < 0.05) in controls. There was no detectable difference with a prolonged maximal physiological AVP concentration (10mU bolus and 50 mU/kg body wt per hr) and papillary solute concentrations were unchanged. When Li-treated rats had been parathyroidectomized (PTX), a significant difference in urine concentration with the low AVP concentration could not be demonstrated when compared to non-PTX control rats. In the isolated papillary collecting duct preparation a medium was used that contained fresh plasma from Li-treated or control rats, both intact and PTX. Experiments using plasma from Li-treated intact rats produced only a 25.4 ± 5.1% increase in diffusional water permeability with the addition of AVP (200 µU/ml) compared to 52.6 ± 9.0% in control rats (P < 0.01). However, when plasma from Li-treated PTX rats was used, the AVP induced increase in water permeability (54.7 ± 11.2%) was not significantly different from that observed in PTX control rats. These studies show that the NDI-like defect in Li-treatment is small and easily overcome by higher concentrations of AVP and suggests that the concentration defect is at least in part due to increased circulating levels of PTH acting as a partial agonist to AVP and thereby inhibiting its hydroosmotic action.

Vasopressin receptors in health and disease. The molecular cloning and characterization of receptors for the nonapeptide hormone family vasopressin-oxytocin was rapidly followed by the identification of mutations in the V 2 receptor gene segregating with the clinical phenotype in more than a hundred families with X-linked nephrogenic diabetes insipidus. Together with the recent cloning of the vasopressin-regulated water channel in the apical membrane of the collecting duct tubule and of the identification of rare autosomal recessive nephrogenic diabetes insipidus patients with mutations in the AQP2 gene, these developments enable carrier detection and early diagnosis of infants with congenital nephrogenic diabetes insipidus.

Expression studies of two vasopressin V2 receptor gene mutations, R202C and 804insG, in nephrogenic diabetes insipidus. Nephrogenic diabetes insipidus (NDI) is a rare X-linked disorder associated with renal tubule resistance to arginine vasopressin (AVP). To understand the mechanisms of AVP resistance underlying this disorder, we have analyzed the vasopressin V2 receptor gene in two unrelated Japanese kindreds with NDI and expressed the mutants to characterize their functional properties. Direct sequencing revealed two V2 receptor gene mutations: a missense mutation from Arg202 to Cys in the third extracellular domain (R202C) and a single base insertion (G) in two consecutive GGG triplets (nucleotide 804 to 809) in the third cytoplasmic domain, resulting in a frame shift with premature termination at codon 258 (804insG). Transient expression study with COS-7 cells showed that R202C mutation reduced both binding affinity (15%) and capacity (30%), while 804insG mutation abolished binding ability. For further evaluation of the binding ability of the R202C mutant, we expressed the mutants in Chinese hamster ovary (CHO) cells. Although the mutant cell lines produced V2 receptor mRNA comparable levels to the wild-type receptor cell lines, R202C mutant cell lines had no binding ability. Our results suggest an introduction of a new cysteine residue in the extracellular domain and a receptor truncation removing one third of the carboxyl terminus could impair ligand binding activity of the V2 receptor through a post-transcriptional mechanism, thereby causing AVP resistance in the NDI patients.

Role of renal prostaglandins during antidiuresis and water diuresis in man. The relationship of renal prostaglandins to antidiuretic hormone action and water diuresis was examined in 13 normal subjects and 2 subjects with diabetes insipidus. Following overnight water deprivation, an oral water load caused a prompt and sustained rise in the rate of urinary PGE2 excretion from 7.7 ± 1.2 to 81.6 ± 26.4 ng/hr (P < 0.0001) in 7 normal subjects. Because the simultaneous increase in urinary excretion of urea was only 17% of the rise in urinary PGE2, passive wash-out of renal PGE2 probably accounts for only a small fraction of the increment in PGE2 excretion. Administration of the antidiuretic hormone analogue DDAVP to 6 normal subjects during sustained water diuresis resulted in a decrease in PGE2 excretion and urine flow rate comparable to that of dehydrated subjects. Thus, PGE2 excretion varied directly with urine flow rate over a wide range of states of hydration in all 13 normal subjects. One patient with central diabetes insipidus and one with nephrogenic diabetes insipidus demonstrated a similar positive correlation of PGE2 excretion rate and urinary flow rate in states of hydration, dehydration, and after administration of DDAVP. In the patient with nephrogenic diabetes insipidus, this relationship of PGE2 excretion rate to urine flow rate was unaffected by DDAVP over a broad range of urine flow rates. Inhibition of prostaglandin synthesis with indomethacin in 6 normal subjects resulted in a significant decline in free water clearance (7.7 ± 1.0 to 4.7 ± 0.9 ml/min, P < 0.001) and an increase in the minimal UOSM (61 ± 4 to 93 ± 19 mOsm/kg, P < 0.01) achieved during water diuresis without a change in creatinine or osmolar clearances. Furthermore, the tightly linked relationship of PGE2 excretion rate to urine flow rate was reduced in 5 of 6 subjects during indomethacin treatment. We conclude that urinary PGE2 excretion varies directly with urine flow rate and is not directly dependent on ADH activity or state of hydration in man. The rise in PGE2 excretion during water diuresis may enhance the excretion of free water since indomethacin treatment blunted free water clearance while suppressing the rise in PGE2 excretion.Rôle des prostaglandines rénales au cours de l'antidiurèse et de la diurèse aqueuse chez l'homme. La relation entre les prostaglandines rénales et l'action de l'hormone antidiurétique et la diurèse aqueuse a été étudiée chez 13 sujets normaux et 2 sujets atteints de diabète insipide. A la suite d'une restriction d'eau pendant la nuit précédant l'étude, une charge d'eau par voie orale a déterminé une augmentation rapide et prolongée du débit urinaire de PGE2 de 7,7 ± 1,2 à 81,6 ± 26,4 ng/hr (P < 0,001) chez 7 sujets normaux. Puisque l'augmentation simultanée de l'excrétion urinaire d'urée était seulement de 17% de l'augmentation de la PGE2 urinaire, un lavage passif de la PGE2 rénale explique probablement une partie seulement de l'augmentation de l'excrétion de PGE2. L'administration d'un analogue de l'hormone antidiurétique, DDAVP, à 6 sujets normaux au cours d'une diurèse aqueuse prolongée a eu pour conséquence une diminution de l'excrétion de PGE2 et du débit urinaire comparable à celle des sujets déshydratés. Ainsi l'excrétion de PGE2 varie directement avec le débit urinaire sur un large éventail d'états d'hydratation chez tous les 13 sujets normaux. Un malade atteint de diabète insipide central et un malade atteint de diabète insipide néphrogénique ont eu une corrélation positive semblable du débit d'excrétion de PGE et du débit urinaire dans les états d'hydratation, de déshydratation, et après l'administration de DDAVP. Chez le malade atteint de diabète insipide néphrogénique, cette relation de l'excrétion de PGE2 au débit urinaire n'a pas été affectée par DDAVP sur un large éventail de valeurs du débit urinaire. L'inhibition de la synthèse de prostaglandine par l'indométhacine chez 6 sujets normaux a eu pour conséquence une diminution significative de la clairance de l'eau libre (7,7 ± 1,0 à 4,7 ± 0,9 ml/mn, P < 0,001) et une augmentation de la valeur minimale de UOSM (61 ± 4 à 93 ± 19 mOsm/kg, P < 0,01) obtenues au cours d'une diurèse aqueuse sans modification de la clairance de la créatinine ou de la clairance osmolaire. De plus, la relation étroite entre l'excrétion de PGE2 et le débit urinaire a été réduite chez 5 des 6 sujets au cours du traitement par l'indométhacine. Nous concluons que l'excrétion urinaire de PGE2 varie directement avec le débit urinaire et n'est pas directement dépendante de l'activité de l'ADH ou de l'état d'hydratation chez l'homme. L'augmentation de l'excrétion de PGE2 au cours de la diurèse aqueuse peut accroître l'excrétion d'eau libre puisque le traitement par l'indométhacine atténue la clairance de l'eau libre en même temps qu'il supprime l'augmentation de l'excrétion de PGE2.

Drug-induced polyuria and polydipsia have been recognized with increasing frequency during the past five years. Several drugs, such as lithium, had been in use for many years without recognition of these toxic side effects, although clinical symptoms appear in almost one out of every ten patients treated with lithium for manic-depressive disorders [1], Other drugs, such as demeclocycline, cause symptoms only after prolonged use or in high concentrations which are not seen in the usual clinical setting [2]. Still other agents, such as the anesthetic methoxyflurane, have had their toxicity recognized almost immediately after their introduction [3, 4]. Finally, some drugs are either too new, such as glipizide [5] and glyburide [6], or have too low orders of toxicity, such as dextropropoxyphene [7], to have had their side effects on water metabolism widely appreciated.

A 15-year-o1d girl was admitted to the New England Medical Center Hospital (NEMCH) for investigation of the hypothalamic-pituitary axis. Four years earlier, an eosinophilic granuloma was discovered in the left mandible and was treated with excision and irradiation (1000 rads). Two years earlier she developed polyuria, and a diagnosis of diabetes insipidus was made at another hospital. She was treated with synthetic lysine vasopressin nasal spray (Diapid) several times a day and noted some symptomatic improvement. Eighteen months before admission, her menstrual periods, which had been normal for the previous 14 months, ceased and she complained of cold intolerance, constipation, and weight gain. She claimed to have grown 0.5 in. over the year prior to admission. She had occasional mild headaches when she did not take her medication but noted no visual disturbances. Family history was unrevealing. Her mother was 61 in. tall.

Effects of demethylchlortetracycline on cellular action of antidiuretic hormone in vitro . Demethylchlortetracycline (DMC) can induce a syndrome of nephrogenic diabetes insipidus in man without affecting other renal functions. Because this pathologic change might be due to interference with the cellular action of antidiuretic hormone (ADH) on the distal nephron, we studied, in vitro , the effect of DMC on some enzymes of the ADH-dependent cyclic AMP (cAMP) system prepared from human renal medulla. Increasing concentrations of DMC produced increasing inhibition of adenylate cyclase basal activity and of the activity stimulated by ADH or by sodium fluoride; at a DMC concentration of 500 µg/ml the inhibition was about 50%. The inhibition appears to be noncompetitive. The same doses of DMC produced only slight inhibition of cAMP diesterase. cAMP-dependent protein kinase contained in cytosol of renal medulla also was inhibited by DMC. Therefore, DMC has the potential to inhibit cAMP formation and the accumulation of cAMP in the renal medulla in response to ADH and also to inhibit cAMP-dependent phosphorylation of proteins in the renal medulla. It is suggested that differential inhibition of these enzymes might be the basis of the unresponsiveness to ADH induced by this drug in vivo . Tetracycline and chlortetracycline had the same effects as DMC in vitro . Additional factors would probably determine why the effect is apparent in vivo only after administration of DMC.

Effects of missense mutations on rat aquaporin-2 in LLC-PK1 porcine kidney cells. Background Mutations in the aquaporin-2 (AQP2) gene have been found in families with nephrogenic diabetes insipidus (NDI), but the pathophysiological mechanisms of how mutant AQP2 causes the disease are still not clear. Methods Wild-type (WT) AQP2 and four mutants—T126M, A147T, R187C, and S216P—were transiently expressed in LLC-PK1 cells. The osmotic water permeability of LLC-PK1 cells expressing AQP2 mutants was determined by stopped-flow light-scattering microphotometry. Cell surface expression, subcellular localization, and effects of vasopressin stimulation were examined by surface biotin labeling and confocal immunohistochemistry. Results The osmotic water permeability (Pf) of cells expressing WT increased significantly after vasopressin treatment, whereas the Pf of cells expressing T126M A147T, R187C, and S216P was not significantly different from that of the control even after vasopressin stimulation. Confocal immunohistochemistry demonstrated distribution of WT and A147T in early/recycling endosomal compartments and vasopressin-responsive translocation and surface expression. In contrast, stainings of T126M, R187C, and S216P were similar to that of Grp78, indicating that these mutants were misassembled and retarded in the endoplasmic reticulum. Conclusion Our results indicated that the intracellular distribution and vasopressin-regulated trafficking of A147T is intact, in contrast to the other three mutants, of which both were impaired. Thus, it is conceivable that the disruption of the AQP2 channel function accounts for the pathogenesis of A147T NDI, whereas trafficking defects account for that of the other types, suggesting that the pathophysiology of AQP2-related NDI is heterogeneous.

AVPR2 variants and V2 vasopressin receptor function in nephrogenic diabetes insipidus. Background The AVPR2 gene encodes the type 2 vasopressin receptor, a member of the vasopressin/oxytocin receptor subfamily of G protein-coupled receptors. Disruption of AVPR2 causes X-linked congenital nephrogenic diabetes insipidus (NDI), yet the functional significance of most gene sequence variations found in association with NDI has not been proven. The large number of naturally occurring AVPR2 mutations constitutes a model system for studying the structure-function relationship of G protein-coupled receptors. This analysis can be aided by examining amino acid sequence variation and conservation among evolutionarily disparate members of the subfamily. Methods Twenty-five new NDI patients were evaluated by DNA sequencing for mutations in AVPR2. Receptors encoded by eighteen NDI alleles were tested for physiologic signaling activity in response to varying concentrations of arginine vasopressin (AVP) in a sensitive cell culture assay. Seventeen amino acid sequences from the vasopressin/oxytocin receptor subfamily were aligned and conserved residues were identified and correlated with the locations of NDI associated variations. Results Twenty-four variant alleles were found among the 25 new patients. Thirteen had no prior family history of expressed NDI. All 18 of the NDI-associated AVPR2 alleles tested for function demonstrated diminished response to stimulation with AVP. Twelve failed to respond at all, whereas six signaled only at high AVP concentrations. Evolutionarily conserved residues clustered in the transmembrane domains and in the first and second extracellular loops, and NDI-associated missense mutations appeared mostly in the conserved domains. Conclusions Sporadic cases are frequent and they usually represent the X-linked rather than the autosomal form of NDI. Genetic and functional testing can confirm this in individual cases. Mutations in this study affecting ligand binding domains tend to retain partial signaling in vitro , whereas those that introduce a charged residue in a transmembrane domain are inactive. The minimal partial signaling observed in cultured cells is unlikely to correlate with clinically significant urine concentrating ability. Other AVPR2 mutations with milder effects on receptor function probably exist, but may not be expressed clinically as typical NDI.

Aquaporin gene delivery to kidney. Background Several aquaporin- (AQP) type water channels are expressed in kidney tubules and microvessels, including AQP1 in proximal tubule, thin descending limb of Henle and vasa recta, AQP2 in collecting duct apical membrane, and AQP3 and AQP4 in collecting duct basolateral membrane. Mice deficient in these aquaporins have distinct phenotypic abnormalities. AQP1 null mice are polyuria and unable to generate a concentrated urine after water deprivation. AQP2-T126M mutant mice and AQP3 null mice manifest nephrogenic diabetes insipidus (NDI) with severe polyuria, whereas AQP4 null mice have only a mild defect in maximal urinary concentrating ability. We reasoned that these mice could serve as useful models for gene replacement because of their predictable and unambiguous phenotypes. Methods In an initial feasibility study, an adenovirus directing the expression of AQP1 was introduced into AQP1 null mice by intravenous infusion. Results At 1 week after adenovirus infusion, AQP1 was seen in many proximal tubules and microvessels. Compared with untreated null mice, the treated mice were able to partially concentrate their urine and lost less weight after water deprivation. However, AQP1 transgene expression and functional correction were lost over 3–5 weeks. Conclusion Although there remain many technical problems to overcome, aquaporin gene replacement has potential applications in hereditary and acquired NDI, and in the transient modulation of renal fluid conservation.

Pathogenesis of nephrogenic diabetes insipidus by aquaporin-2 C-terminus mutations. Background We previously reported three aquaporin-2 (AQP2) gene mutations known to cause autosomal-dominant nephrogenic diabetes insipidus (NDI) ( Am J Hum Genet 69:738, 2001). The mutations were found in the C-terminus of AQP2 (721delG, 763 to 772del, and 812 to 818del). The wild-type AQP2 is a 271 amino acid protein, whereas these mutant genes were predicted to encode 330 to 333 amino acid proteins due to the frameshift mutations leading to the creation of a new stop codon 180 nucleotides downstream. The Xenopus oocyte expression study suggested that the trafficking of the mutant AQP2s was impaired. Methods To determine the cellular pathogenesis of these NDI-causing mutations in mammalian epithelial cells, Madin-Darby canine kidney (MDCK) cells were stably transfected with the wild-type AQP2, or the 763 to 772del mutant AQP2, or both. Cells were grown on the membrane support to examine the localization of AQP2 proteins by immunofluorescence microscopy. Results Confocal immunofluorescence microscopy showed that the wild-type AQP2 was expressed in the apical region, whereas the mutant AQP2 was apparently located at the basolateral region. Furthermore, the wild-type and mutant AQP2s were colocalized at the basolateral region when they were cotransfected, suggesting the formation of mixed oligomers and thereby mistargeting. Conclusion Mixed oligomers of the wild-type and the 763 to 772del mutant AQP2s are mistargeted to the basolateral membrane due to the dominant-negative effect of the mutant. This defect is very likely to explain the pathogenesis of autosomal-dominant NDI. The mistargeting of the apical membrane protein to the basolateral membrane is a novel molecular pathogenesis of congenital NDI.