Methods and compositions for treating nephrogenic diabetes and insipidus   

This application claims priority to U.S. Provisional Application 60/621,910 filed on Oct. 25, 2004 by Bellamkonda K. Kishore, Noel Carlson, Donald Kohan, and Raoul Nelson, and this application is incorporated herein in its entirety by reference.

This invention was made possible with the facilities and resources at the VA Salt Lake City Health Care System. This invention was also made with government support under federal grants DK61183 DK58953, and 53990 awarded by the NIH. Therefore, the United States Government may have certain rights in this invention.

SUMMARY

As embodied and broadly described herein, the disclosed compositions and methods, in one aspect, relate to the treatment of nephrogenic diabetes insipidus (NDI). This application also relates to the use of P2Y2 agonists as diuretics. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the compositions and methods and together with the description, serve to explain the principles of the compositions and methods.

FIG. 1 shows water balance disorders associated with altered expression of vasopressin-regulated collecting duct water channel AQP2. As shown here, AQP2 protein expression is reduced, sometimes dramatically, in a wide variety of hereditary and acquired forms of diabetes insipidus characterized by varying degrees of polyuria. On the other hand, water retention conditions like heart failure and pregnancy are associated with increased expression of AQP2 protein. (Nielsen et al, 1999).

FIG. 2 shows schematic representation of the two mutually opposing signaling pathways and the corresponding membrane receptors involved in the regulation of osmotic water permeability of medullary collecting duct principal cell. The scheme also illustrates the points where the two signaling pathways interact (Schwiebert and Kishore, 2001).

FIG. 3 shows the release of PGE2 by the agonist stimulation of P2Y2 receptor. FIG. 3A shows time-course of release of PGE2 by the agonist stimulation of P2Y2 receptor in rat IMCD preparations in vitro. Freshly prepared IMCD-enriched fractions were incubated at 37° C. under physiological conditions in the absence (vehicle, ∘) or presence (•) of 100 μM of ATPγS for different periods of time. PGE2 released into the medium was assayed by EIA and normalized to the protein content of the incubations. Results are expressed as mean±SEM of triplicate incubations. *significantly different from the corresponding vehicle alone incubations. **significantly different from the corresponding 0, 5 and 10 min values. FIG. 3B shows concentration-response curve for the release of PGE2 by the agonist stimulation of P2Y2 receptor in rat IMCD preparations in vitro. Freshly prepared IMCD-enriched fractions were incubated at 37° C. for 10 min with increasing concentrations of ATPγS (0-100 μM). PGE2 released into the medium was assayed by EIA and normalized to the protein content of the incubations. Results are mean±SEM of triplicate incubations. *significantly different from the 0 μM ATPγS incubations.

FIG. 4 shows purinergic and prostanoid interactions in the inner medulla of hydrated and dehydrated rats. Animal model: Hydration and dehydration of rats was achieved by either adding sucrose (600 mM) to drinking water or by depriving drinking water, respectively, for 2 days prior to euthanasia. Control rats received plain tap water. All rats had free access to standard rat chow. Urine samples were collected from rats by housing them in individual metabolic cages during the last 24 hours of experimental period. Twenty-four hour urine volumes and osmolality were determined. Hydrated rats had high urine output of very dilute urine and dehydrated rats had low output of concentrated urine. Panels A-C: Altered protein abundance of P2Y2 receptor in the inner medulla of hydrated and dehydrated rat kidneys. Whole tissue homogenates were prepared, solubilized and immunoblotted for P2Y2 protein. The P2Y2 receptor antibody identifies two sets of immunoreactive bands in the kidney (Kishore et al, 2000a) (panels A & B). Both 47 kDa and 105 kDa bands are specific, as these were ablated by pre-adsorption of the antibody with the immunizing peptide. Blots were digitized, band densities determined, and expressed as percent of mean values in controls (panel C). Numerical results are expressed as mean±SEM. *significantly different from the corresponding band in the other group. Panel D: Urinary excretion of PGE2 metabolites in control, hydrated and dehydrated rats. Groups of male rats (N=3 per group) were subjected to hydration and dehydration. Twenty-four hour urine samples were collected and assayed for PGE2 metabolite, using a commercial kit (Cayman Chemical Co, Ann Arbor, Mich.). This assay converts all the immediate PGE2 metabolites to a single, stable derivative that could be easily quantified by EIA. Measured urinary excretion of PGE2 metabolite values (ng/24 hours) are expressed as percent of mean values in the control group. *significantly different from the other two groups. ** significantly different from the control group. Panel E: In vitro IMCD response to purinergic-stimulated PGE2 release in control, hydrated and dehydrated rats. Groups of rats (N=3 per group) were subjected to dehydration and hydration. Inner medullae from each group were pooled, and fractions enriched in IMCD were prepared from these pools by collagenase and hyaluronidase digestion (Welch et al, 2003). The pooled IMCD preparations from each group was divided into two sets of tubes (N=4 per set). One set served as vehicle control (baseline value), while the other set of preparations was challenged with 50 μM of ATPγS (a non-hydrolyzable agonist of P2Y2 receptor) for 20 min at 37° C. The reaction was arrested by adding chilled incubation buffer. Samples were centrifuged to pellet IMCD, and the PGE2 concentration in the supernatants was assayed by a commercial EIA kit and normalized to the protein content of the incubations (ng/mg protein). ATPγS-stimulated release of PGE2 in each group was computed as percent increase over the respective vehicle controls (baseline values). Results are expressed as mean±SEM. *significantly different from the other two groups. The PGE2 release from the IMCD of dehydrated rats is significantly lower than that of controls.

FIG. 5 shows characterization of rat model of lithium-induced NDI. Panel A: Body weights of rats fed control or lithium-added diets. Panel B: Water intake in rats fed control or lithium-added diets. Panel C: Twenty-four hour urine volumes in rats fed control or lithium-added diets. Panel D: Urine osmolalities in rats fed control or lithium-added diets. Panel E: Protein abundance of AQP2 water channel in the inner medulla of rats fed control or lithium-added diets, as determined by Western blotting. Panel F: Densitometry of AQP2 protein bands in rats fed control or lithium-added diets. Results are expressed as mean±SEM. *significantly different from the corresponding value in control diet group.

FIG. 6 shows protein abundance and mRNA expression of P2Y2 receptor in inner medulla (panel A & B), and in vitro IMCD response to purinergic-stimulated PGE2 release (panel C) in rats fed control or lithium-added diets for 21 days. Inner medullae from control or lithium diet fed rats were pooled separately, and fractions enriched in IMCD were prepared from these pools by collagenase and hyaluronidase digestion. Experiments were carried out as described for hydrated and dehydrated rats in FIG. 4. ATPγS-stimulated release of PGE2 in each group was computed as percent increase over the respective vehicle controls (baseline values). The mean increase in P2Y2-stimulated release of PGE2 in lithium group is ˜2-fold higher as compared to the mean increase in controls. Results are expressed as mean±SEM. *significantly different from the control group.

FIG. 7 shows expression of cytosolic phospholipase A2 (cPLA2; panel A), cyclooxygenase-1 (COX-1; panel B) and cyclooxygenase-2 (Cox-2; panel C) messenger RNA in the renal inner medulla of rats fed control or lithium added diets for 21 days. Messenger mRNA extraction, purification, reverse transcription, real-time PCR amplifications and computation of results were carried out as described for P2Y2 receptor in FIG. 6 legend. The sequences of primer pairs used in these amplifications are shown in Table 6. The expression of cPLA2, COX-1 and COX-2 genes was normalized to the expression of housekeeping gene β-actin (Relative Expression), and computed as percent or mean values in the control diet group. Results are mean±SEM. *significantly different from the control diet group. Lithium diet fed rats showed approximately 2-, 3- and 1.5-fold increase in mRNA expression of cPLA2, COX-1 and COX-2 as compared to the control diet fed group.

FIG. 8 shows the characterization of rat model of NDI induced by bilateral ureteral obstruction (BUO) and release. Panel A: Twenty-four hour urine volumes in sham operated and BUO rats. *significantly different from the corresponding sham operated values by ANOVA followed by Tukey-Kramer Multiple Comparison Test; **significantly different from the corresponding sham operated values by unpaired t test. Panel B: Urine osmolalities in sham operated and BUO rats. *significantly different from the corresponding sham operated values. The decrease in mean urine osmolality in BUO rats on day 12 did not attain statistical significance (P=0.112) due to the large variation in sham operated rats and small sample size. Panel C: Water intake in sham operated rats (pooled values from day 0, 7 and 12) and BUO rats at day 0, 7 and 12. *significantly different from the values in BUO day 0 and sham operated groups. Panel D: Urinary excretion of PGE2 metabolite in sham operated and BUO rats measured on day 12 urine samples. *significantly different from the sham operated values. Panel E: Protein abundance of AQP2 water channel in the inner medulla of sham operated and BUO rats euthanized on day 12. Panel F: Densitometry of AQP2 protein bands in sham operated and BUO rats. *significantly different from the corresponding band density in sham operated group. Results are expressed as mean±SEM. The number in parenthesis indicates the number of animals examined. Since the surgical procedures were performed on small batches of animals on each time, some of the urine samples could not be collected for analysis. Day 0 represents the period just prior to the first surgical operation performed to obstruct the ureters.

FIG. 9 shows protein abundance (panel A) and mRNA expression (panel B) of P2Y2 receptor in the inner medulla, and in vitro IMCD response to purinergic-stimulated PGE2 release (panel C) in sham operated and BUO rats. Sham operated (N=2) and BUO (N=3) rats were euthanized on day 7. Inner medullae from sham operated and BUO rats were pooled separately, and fractions enriched in IMCD were prepared from these pools by collagenase and hyaluronidase digestion. Experiments were carried out as described for hydrated and dehydrated rats in FIG. 4. ATPγS-stimulated release of PGE2 in group was computed as percent increase over the respective vehicle controls (baseline values). Results are expressed as mean±SEM of four incubations from pooled IMCD in each group. *significantly different from the sham operated group. The mean increase in P2Y2-stimulated release of PGE2 in BUO rats is about 4.3-fold higher as compared to the mean increase in sham operated controls.

FIG. 10 shows expression of cytosolic phospholipase A2 (cPLA2; panel A), cyclooxygenase-1 (COX-1; panel B) and cyclooxygenase-2 (Cox-2; panel C) mRNA in the renal inner medulla of sham operated and BUO rats. Messenger mRNA extraction, purification, reverse transcription, real-time PCR amplifications and computation of results are carried out as described for P2Y2 receptor in FIG. 6 legend. The sequences of primer pairs used in these amplifications are shown in Table 6. The expression of cPLA2, COX-1 and COX-2 genes was normalized to the expression of housekeeping gene β-actin (Relative Expression), and computed as percent or mean values in the control diet group. Results are mean±SEM. *significantly different from the sham operated group. BUO rats showed approx. 2-, and 3-fold increase in mRNA expression of COX-1 and COX-2 as compared to the sham operated group.

FIG. 11 shows the sequence of intracellular events leading from vasopressin V2 receptor on the basolateral membrane to the insertion of AQP2 water channels into the apical membrane of collecting duct principal cell. The potential pre-cAMP formation sites that can be disrupted are (i) activation of inhibitory G protein (Gi) by the increased activity of PKC brought about by diacylglycerol (DAG) formed as a result of stimulation of the PI signaling pathway by various autocrine and paracrine agents (PGE2, ATP/UTP or endothelin), and (ii) decreased activity of adenylyl cyclase (AC) isoforms 5 and 6 expressed in the medullary collecting duct. The potential post-cAMP formation sites which can be disrupted are (i) rapid hydrolysis of cAMP by phoshodiesteases (isofoms III and IV) expressed in medullary collecting duct, and (ii) decreased activity of PKA, resulting in decreased protein phosphorylation and membrane insertion of AQP2

FIG. 12 shows the determination of the degree of phosphorylation of cPLA2. It is achieved by prolonged low voltage PAGE electrophoresis of solubilized tissue homogenates, so that the native and phosphorylated species clearly separate, and then transferring the separated proteins to a nitrocellulose membrane and immunoblotting with a specific antibody to cPLA2. Figure shows native (red arrow) and phoshorylated cPLA2 (blue arrow) in inner medullary homogenates of 5 normal rats separated in our laboratory using PAGE and immunoblotting.

FIG. 13 shows the effect of depletion of extracellular ATP by apyrase treatment on the urine flow (left panel) and on and P2Y2 receptor-stimulated ex vivo PGE2 release by IMCD (right panel).

FIG. 14 shows dependency on the COX-2 activity of P2Y2 receptor-mediated PGE2 release by IMCD in lithium-induced NDI (right panel) and the lack of such dependency on the activity of COX-2 in sucrose-water induced polyuria (left panel).

FIG. 15 shows the proposed models for the interaction among vasopressin (AVP), purinergic (ATP), and prostanoid (PGE2) systems in medullary collecting duct principal cell under normal conditions (left) and how they are deranged in acquired NDI (right). (−) and (+) signs denote inhibition and stimulation respectively. X marks indicate blocked pathways. Larger size of the arrows indicates accentuation of pathways. The letters A, B, C, D and E are keyed to the explanation in Example 9.

DETAILED DESCRIPTION

The present compositions and methods can be understood more readily by reference to the following detailed description and the Examples included therein and to the Figures and their previous and following description.

Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that the compositions and methods are not limited to specific synthetic methods, specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed then “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed.

“Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The terms “higher,” “increases,” “elevates,” or “elevation” refer to increases above basal levels, e.g., as compared to a control. The terms “low,” “lower,” “reduces,” or “reduction” refer to decreases below basal levels, e.g., as compared to a control. For example, basal levels are normal in vivo levels prior to, or in the absence of, addition of an agent.

“Kidney cells” include all renal tubular epithelial cells, renal cortical tubules, glomerular cells, mesangial cells, interstitial cells, collecting duct prinicipal cells, and intercalated cells of the kidney.

The term “diabetes insipidus” includes, but is not limited to, any disease of the kidneys such as neurogenic, also known as central, hypothalamic, pituitary, or neurohypophyseal diabetes; nephrogenic, also known as vasopressin-resistant; gestanic; and dipsogenic diabetes.

The term “test compound” is defined as any compound to be tested for its ability to interact with a selected cell, e.g., a P2Y antagonist. Examples of test compounds include, but are not limited to, suramin, acid blue 129, and acid blue 80. Also, “test components” include drugs, molecules, and compounds that come from combinatorial libraries where thousands of such ligands are screened by drug class.

The terms “control levels” or “control cells” are defined as the standard by which a change is measured, for example, the controls are not subjected to the experiment, but are instead subjected to a defined set of parameters, or the controls are based on pre- or post-treatment levels.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

Acquired nephrogenic diabetes insipidus (NDI), which is relatively common, comprises several clinical conditions, such as lithium-induced nephropathy, hypokalemic nephropathy, hypercalcemia, and post-obstructive uropathy. The hallmark of these conditions is low protein levels of vasopressin-regulated water channel AQP2 in the medullary collecting duct, in the presence of normal or elevated circulating levels of arginine vasopressin (AVP). In both human patients and in experimental animals with acquired NDI, the production of renal prostaglandins such as PGE2, is increased. PGE2, by virtue of its ability to antagonize AVP-stimulated water permeability via retrieval of AQP2 water channels from the apical membrane of inner medullary collecting duct (IMCD), is involved in the development of polyuria of acquired NDI. Accordingly, inhibition of PGE2 synthesis by the administration of indomethacin was shown to ameliorate the polyuria of acquired NDI. In rat IMCD, agonist stimulation of P2Y2 purinergic (nucleotide) receptor results in production and release of PGE2 (Welch et al, 2003), and this response is markedly enhanced in hydrated polyuric rats (Sun et al, 2004). It has been shown that the purinergic-mediated PGE2 release in IMCD is also markedly enhanced in acquired NDI induced by lithium (Li) administration or by bilateral ureteral obstruction (BUO) and release. And this is associated with significant increases in mRNA expression of cyclooxygenases-1 and -2 in inner medulla of acquired NDI rats.

Diabetes insipidus (DI) causes considerable morbidity and inconvenience to the patients. Patients with DI, especially those critically ill, are at higher risk of dehydration, hypematremia, alterations in the level of consciousness, and hemodynamic instability from hypovolemia, for example (Bell, 1994). Acquired nephrogenic diabetes insipidus (NDI), the more common form of NDI, can occur at any age. The most common cause of acquired NDI is lithium administration for the treatment of bipolar disorders. Other drugs that are capable of inducing acquired NDI are colchicine, methoxyflurane, amphotericin B, gentamicin, loop diuretics, and demeclocycline, for example. In addition to drugs, acquired NDI can also occur as a result of certain diseases. These include, but are not limited to chronic kidney diseases, hypokalemia, hypercalcemia, sickle cell disease, ureteral obstruction (obstructive uropathy), and low protein diet. The hallmark of these conditions, as documented in animal models, is low protein abundance of AVP-regulated water channel AQP2 in the medullary collecting duct in the face of normal or elevated circulating levels of AVP (Nielsen et al, 1999; FIG. 1). Thus, in these conditions, it appears that the inherent defect lies in the collecting duct.

The collecting duct system, which expresses AQP2, AQP3 and AQP4 water channels, accounts for the absorption of 15-20% of the filtered water. This is precisely regulated by AVP, and thus it is crucial for the conservation of body water and excretion of concentrated urine. AQP2 water channel, expressed on the apical plasma membrane and on sub-apical vesicles of collecting duct principal cells, is regulated by AVP. AVP, acting through its V2 receptor, a G protein-coupled receptor, on the collecting duct principal cells, activates membrane bound adenylyl cyclase (AC) to produce cAMP as a second messenger (FIG. 2). The cellular effects of cAMP are believed to be connected to the activation of protein kinase A (PKA), which phosphorylates various key proteins. AVP has both short- and long-term effects on the collecting duct water permeability. As depicted in FIG. 2, the short-term regulation (in the time frame of few to several minutes) of collecting duct water permeability by AVP involves the translocation of AQP2 water channels from a pool of subapical vesicles to the apical plasma membrane (Nielsen et al, 1995). The apical plasma membrane is the rate-limiting barrier for the transepithelial water movement, as AQP3, AQP4 are constitutively expressed on the basolateral domain of the collecting duct principal cells under normal conditions (FIG. 2). The long term-regulation (within the time frame of several hours to days) of collecting duct water permeability involves a parallel increase in the absolute amount of AQP2 mRNA and protein (Agre, 2000; Krane and Kishore, 2003). Water deprivation and vasopressin stimulation both increase AQP2 protein expression and apical membrane targeting (Nielsen et al, 1993; DiGiovanni et al, 1994; Kishore et al, 1996). cAMP is capable of stimulating AQP2 gene transcription by acting through CRE and AP1 sites in the AQP2 promoter (Hozawa et al, 1996; Yasui et al, 1997; Matsumura et al, 1997). cAMP activation of AQP2 gene likely occurs by phosphorylation of CREB (CRE binding protein) and the ability of phosphorylated CREB to activate AQP2 gene transcription via binding to CRE sites in the AQP2 promoter. cAMP activation of AQP2 gene could also occur by the induction of c-Fos expression and c-Fos activation of AQP2 transcription via the API site in the AQP2 promoter.

Apart from AVP, a variety of autocrine and paracrine agents, such as PGE2, endothelin and extracellular nucleotides (ATP/UTP), also regulate the collecting duct water permeability. Acting via their respective receptors and the accompanying phosphoinositide signaling pathway these agents decrease the osmotic water permeability of the collecting duct (FIG. 2), even in the presence of AVP (Nadler et al, 1992; Kohan and Hughe, 1993; Kishore et al, 1995; Roman and Lechene, 1981; Rouch and Kudo, 2000). Thus, in the collecting duct, cyclic AMP and phosphoinositide systems are mutually opposing signaling pathways (Teitelbaum, 1992). Diacylglycerol (DAG) formed as a result of stimulation of PI signaling pathway stimulates the activity of PKC, which in turn induces the activity of Gi (inhibitory G protein) associated with the V2 receptor complex. Activation of Gi uncouples the signal from V2 receptor to adenylyl cyclase (AC), resulting in decreased cellular cAMP levels. Activation of PI signaling pathway also results in the stimulation of specific phoshodiesterases (PDEs) through the calcium-calmodulin (CaM) pathway. These PDEs rapidly hydrolyze cAMP and thus reduce the water permeability of the collecting duct as demonstrated in DI+/+mice, which exhibit constitutively active cAMP-PDE (PDE type IV) (Homma et al, 1991; Frøkiær et al, 1999)

Both in human patients and in laboratory animals, lithium-induced NDI and post-obstructive uropathy are associated with increased production and excretion of PGE2 in urine. And administration of indomethacin ameliorated these polyuric conditions, indicating that PGE2 is involved in the genesis of polyuria (Laszlo et al, 1980; Fradet et al, 1980, 1988; Sugawara et al, 1998). PGE2 is a major prostanoid in the kidney and it interacts with four G protein-coupled E-prostanoid receptors designated EP1, EP2, EP3 and EP4. Through these receptors, PGE2 modulates renal hemodynamics and salt and water excretion (Breyer and Breyer, 2000). PGE2 has an antagonistic effect on AVP-stimulated collecting duct water permeability (Nadler et al, 1992; Han et al, 1994), and molecular mechanisms of this effect of PGE2 on AVP-stimulated water permeability in renal collecting duct have been shown. Using ex-vivo preparations of renal medulla, Zelenina et al (2000) have demonstrated that agonist stimulation of EP3 prostanoid receptor causes retrieval of AQP2 water channels from the apical membrane, thus reducing the abundance of AQP2 protein in the apical membrane, the rate-limiting barrier in the transepithelial water movement in the collecting duct.

P2Y2 receptor is a G protein-coupled nucleotide receptor, linked to phosphoinositide signaling pathway. The agonist potency order of P2Y2 receptor is typically UTP=ATP>ATPγS>2MeS-ATP>α,β-MeATP. Since agonist activation of this receptor results in the mobilization of intracellular Ca2+ (Ecelbarger et al., 1994), activation of P2Y2 receptor in medullary collecting duct can result in the down-regulation of AVP-stimulated water permeability. Using a model of in vitro microperfused terminal inner medullary collecting duct (IMCD) of rat, it was demonstrated that ATP or UTP, but not ADP (a non-agonist), decreased the AVP-stimulated osmotic water permeability (Pf) in a reversible fashion. Studies using a non-hydrolyzable cAMP analog or forskolin or calphostin C(PKC inhibitor) revealed that the mechanism of this inhibition involves a pre-cAMP formation site, probably the inhibitory G protein (Gi) (Kishore et al., 1995).

In order to establish the molecular expression in IMCD, and to study its regulation in pathophysiological conditions, molecular tools were developed (antibody, primers and cDNA probe) to detect the protein and mRNA of P2Y2 receptor at cellular and tissular levels in the kidney (Kishore et al., 2000a). The rat P2Y2 receptor cDNA cloned from type II alveolar cells, which were used to design an antibody, primers and cDNA probe, has an open reading frame of 1125 base pairs (359-1438 bp) with no introns (Rice et al, 1995). The open reading frame encodes a putative protein of 374 amino acid residues with a predicted molecular weight of 42,275 Daltons in the unglycosylated state (Rice et al, 1995). The protein contains seven transmembrane-spanning domains, characteristic of G protein-coupled receptors. To authenticate the specificity and reliability of the tools, they were also tested on lung tissue, where the expression and function of P2Y2 receptor were well known. RT-PCR and Northern analysis showed expression of P2Y2 mRNA in both lung and kidney. RT-PCR on microdissected collecting duct segments demonstrated P2Y2 receptor mRNA expression in collecting ducts. Immunoblots using a C-terminal peptide-derived polyclonal antibody to P2Y2 receptor showed that IMCD expresses two distinct and specific products (47 and 105 kDa), and account for the majority of the receptor expression in the inner medulla. Immunoperoxidase labeling on cryosections showed localization of P2Y2 receptor protein in the apical and basolateral domains of IMCD principal cells in the kidney, and on Clara cells and goblet cells in terminal respiratory bronchioles (Kishore et al, 2000a).

A rise in intracellular calcium, such as the one that occurs following agonist stimulation of P2Y2 receptor, is known to be frequently associated with release of arachidonic acid in several tissues or cells. In most of these tissues or cells, especially those of nonendothelial nature, the predominant prostanoid produced was PGE2. Thus, it was hypothesized that the agonist stimulation of P2Y2 receptor in IMCD should also result in production and release of PGE2. To test this hypothesis, experiments were conducted on freshly prepared rat IMCD fractions, and the effect of activation of P2Y2 receptor on the release of PGE2 was examined. The results show that unstimulated IMCD released significant, amounts of PGE2. Agonist activation of P2Y2 receptor by ATPγS enhanced release of PGE2 from IMCD in a time- and concentration-dependent fashion (FIG. 3). Furthermore, purinergic-stimulated release of PGE2 was completely blocked by non-specific COX inhibitors. Differential COX inhibition studies revealed that purinergic-stimulated release of PGE2 was more sensitive to a COX-1 specific inhibition than COX-2 specific inhibition (Welch et al. 2003). Because PGE2 is known to affect transport of water, salt, and urea in IMCD (Nadler et al, 1992; Roman and Lechene, 1981; Rouche and Kudo, 2000), the observed interaction of purinergic system with the prostanoid system in IMCD can modulate handling of water, salt, and urea by IMCD and, thus constitutes a complex AVP-independent regulatory mechanism. Thus, the purinergic regulation of medullary collecting duct function extends beyond the direct modulation of AVP-stimulated water permeability.

Potato apyrase (EC 3.6.1.5) is a soluble NTPDase, exhibiting both ATPase and ADPase activities. It is non-toxic and safe to administer either intravenously or intraperitoneally. As documented in FIG. 13, apyrase treatment from day 7 to 14 in lithium-fed rats (i) prevented further increase in the urine flow induced by lithium, and (ii) significantly decreased the P2Y2 receptor-stimulated PGE2 release by IMCD as compared to the apyrase-untreated and lithium-fed rats. Apyrase treatment did not change the urine output or P2Y2-mediated PGE2 release by IMCD in control rats fed with regular diet. These observations showed that apyrase has the ability to decrease the PGE2 formation in acquired NDI by novel means other than the direct inhibition of the activities of cyclooxygenases.

Apyrase can be used for blocking the purinergic signaling, especially following xenograft. Apyrase can also be optimized for administration alone or in combination with specific P2Y2 receptor antagonists to achieve the best possible effect in controlling polyuria and the ensuing hypernatremia.

1. Purinergic Receptors

Several cell membrane receptors, which preferentially bind extracellular nucleotides (ATP/UTP/ADP), and their analogues have been identified, cloned and characterized. There receptors, collectively known as extracellular nucleotide receptors or purinergic receptors have been classified based on their molecular biology, biological actions and pharmacology. Broadly they are divided into P2Y and P2X families. (P1 receptors are not nucleotide receptors; they are adenosine receptors). The P2X receptors are ionotrophic ATP-gated channels that open up to allow small molecules to enter into the cells. Purinergic regulation of renal function encompasses glomerular hemodynamics, microvascular function, tubuloglomerular feedback, tubular transport, renal cell growth and apoptosis for example (Schwiebert and Kishore, 2001; Inscho, 2001).

There are two main families of purine receptors, adenosine or P1 receptors, and P2 receptors, recognizing primarily ATP, ADP, UTP, and UDP (Table 1). Adenosine/P1 receptors couple to G proteins and have been further subdivided, based on molecular, biochemical, and pharmacological evidence into four subtypes, A1, A2A, AB, and A3. In contrast, P2 receptors divide into two families of ligand-gated ion channels and G protein-coupled receptors termed P2X and P2Y receptors, respectively. For example, Table 1 sets forth seven mammalian P2X receptors (P2×7) and five mammalian P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) which have been cloned and characterized.

Adenosine/P1 receptors P2 receptors Natural Adenosine ATP, ADP, UTP, UDP, Adenine ligands dinucleotides Subgroup — P2X P2Y Type G protein-coupled Ion channel G protein-coupled Nonselective pore Subtypes A1, A2A, A2B, A3 P2X1-7, P2Xn P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2YADP (or P2T) Uridine nucleotide-specific

P2Y receptors are purine and pyrimidine nucleotide receptors that are coupled to G proteins. Most P2Y receptors act via G protein coupling to activate PLC leading to the formation of IP3 and mobilization of intracellular Ca2+. Coupling to adenylate cyclase by some P2Y receptors has also been described. The response time of P2Y receptors is longer than that of the rapid responses mediated by P2X receptors because it involves second-messenger systems and down stream mediators mediated by G protein coupling. Five mammalian P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) have been cloned, and functionally characterized and show distinct pharmacological profiles (Table 2).

TABLE 2 Cloned P2Y receptors Acession cDNA library Receptor number source Agonist activity References P2Y1 Human brain 2MeSATP > ATP  UTP Schachter et al., 1996 (362 S81950 Human prostate 2MeSATP > ATP = ADP Janssens et al., 1996 amino and ovary acids Z49205 Human Léon et al., 1995, 1997

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