Protein phosphatase 1 inhibitor-1 deficiency reduces phosphorylation of renal NaCl cotransporter and causes arterial hypotension.

The thiazide-sensitive NaCl cotransporter (NCC) of the renal distal convoluted tubule (DCT) controls ion homeostasis and arterial BP. Loss-of-function mutations of NCC cause renal salt wasting with arterial hypotension (Gitelman syndrome). Conversely, mutations in the NCC-regulating WNK kinases or kelch-like 3 protein cause familial hyperkalemic hypertension. Here, we performed automated sorting of mouse DCTs and microarray analysis for comprehensive identification of novel DCT-enriched gene products, which may potentially regulate DCT and NCC function. This approach identified protein phosphatase 1 inhibitor-1 (I-1) as a DCT-enriched transcript, and immunohistochemistry revealed I-1 expression in mouse and human DCTs and thick ascending limbs. In heterologous expression systems, coexpression of NCC with I-1 increased thiazide-dependent Na(+) uptake, whereas RNAi-mediated knockdown of endogenous I-1 reduced NCC phosphorylation. Likewise, levels of phosphorylated NCC decreased by approximately 50% in I-1 (I-1(-/-)) knockout mice without changes in total NCC expression. The abundance and phosphorylation of other renal sodium-transporting proteins, including NaPi-IIa, NKCC2, and ENaC, did not change, although the abundance of pendrin increased in these mice. The abundance, phosphorylation, and subcellular localization of SPAK were similar in wild-type (WT) and I-1(-/-) mice. Compared with WT mice, I-1(-/-) mice exhibited significantly lower arterial BP but did not display other metabolic features of NCC dysregulation. Thus, I-1 is a DCT-enriched gene product that controls arterial BP, possibly through regulation of NCC activity.

Overexpression of pendrin in intercalated cells produces chloride-sensitive hypertension.

Inherited and acquired disorders that enhance the activity of transporters mediating renal tubular Na(+) reabsorption are well established causes of hypertension. It is unclear, however, whether primary activation of an Na(+)-independent chloride transporter in the kidney can also play a pathogenic role in this disease. Here, mice overexpressing the chloride transporter pendrin in intercalated cells of the distal nephron (Tg(B1-hPDS) mice) displayed increased renal absorption of chloride. Compared with normal mice, these transgenic mice exhibited a delayed increase in urinary NaCl and ultimately, developed hypertension when exposed to a high-salt diet. Administering the same sodium intake as NaHCO3 instead of NaCl did not significantly alter BP, indicating that the hypertension in the transgenic mice was chloride-sensitive. Moreover, excessive chloride absorption by pendrin drove parallel absorption of sodium through the epithelial sodium channel ENaC and the sodium-driven chloride/bicarbonate exchanger (Ndcbe), despite an appropriate downregulation of these sodium transporters in response to the expanded vascular volume and hypertension. In summary, chloride transport in the distal nephron can play a primary role in driving NaCl transport in this part of the kidney, and a primary abnormality in renal chloride transport can provoke arterial hypertension. Thus, we conclude that the chloride/bicarbonate exchanger pendrin plays a major role in controlling net NaCl absorption, thereby influencing BP under conditions of high salt intake.

Genetic ablation of aquaporin-2 in the mouse connecting tubules results in defective renal water handling.

Body water balance is regulated via the water channel aquaporin-2 (AQP2), which is expressed in the renal connecting tubule (CNT) and collecting duct (CD). The relative roles of AQP2 in the CNT and CD are not fully understood. To study the role of AQP2 in the CNT we generated a mouse model with CNT-specific AQP2 deletion (AQP2-CNT-knockout (KO)). Confocal laser scanning microscopy and immunogold electron microscopy demonstrated an absence of AQP2 in the CNT of AQP2-CNT-KO mice. Twenty-four hour urine output was significantly increased (KO: 3.0 ± 0.3 ml (20 g body weight (BW))(-1); wild-type (WT): 1.9 ± 0.3 ml (20 g BW)(-1)) and urine osmolality decreased (KO: 1179 ± 107 mosmol kg(-1); WT: 1790 ± 146 mosmol kg(-1)) in AQP2-CNT-KO mice compared with controls. After 24 h water restriction, urine osmolality was still significantly lower in AQP2-CNT-KO mice (KO: 2087 ± 169 mosmol kg(-1); WT: 2678 ± 144 mosmol kg(-1)). A significant difference in urine osmolality between groups before desmopressin (dDAVP) (KO: 873 ± 129 mosmol kg(-1); WT: 1387 ± 163 mosmol kg(-1)) was not apparent 2 h after injection, with urine osmolality increased significantly in both groups (KO: 2944 ± 41 mosmol kg(-1); WT: 3133 ± 66 mosmol kg(-1)). Cortical kidney fractions from AQP2-CNT-KO mice had significantly reduced AQP2, with no compensatory changes in sodium potassium chloride cotransporter (NKCC2), AQP3 or AQP4. Lithium chloride treatment increased urine volume and decreased osmolality in both WT and AQP2-CNT-KO mice. After 8 days of treatment, the AQP2-CNT-KO mice still had a significantly higher urine volume and lower urine osmolality, suggesting that the CNT does not play a significant role in the pathology of lithium-induced nephrogenic diabetes insipidus. Our studies indicate that the CNT plays a role in regulating body water balance under basal conditions, but not for maximal concentration of the urine during antidiuresis.

Vasopressin inhibits apoptosis in renal collecting duct cells.

The peptide hormone arginine vasopressin (AVP) plays a critical role in regulating salt and water transport in the mammalian kidney. Recent studies have also demonstrated that AVP can promote cell survival in neuronal cells through V1 receptors. The current study addresses whether AVP can inhibit apoptosis in kidney collecting duct cells via V2 receptors and also explores the downstream signaling pathways regulating this phenomenon. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling analysis and caspase cleavage assays demonstrated that 1-desamino-8-d-arginine vasopressin (dDAVP) inhibited apoptosis induced by various agents (staurosporine, actinomycin D, and cycloheximide) in cultured mouse cortical collecting duct cells (mpkCCD). Incubation with dDAVP also inhibited apoptosis induced by the phosphatidylinositol 3-kinase (PI3K) pathway inhibitor LY294002, suggesting that the antiapoptotic effects of dDAVP are largely independent of PI3K signaling. The V2 receptor antagonist SR121463 completely abolished the antiapoptotic effects of dDAVP. In addition, incubation with 8-cpt-cAMP, a cell-permeable analog of cAMP, reproduced the antiapoptotic effects of dDAVP. Both dDAVP and 8-cpt-cAMP increased phosphorylation of proapoptotic Bcl-2 family members Bad and Bok. Bad phosphorylation at Ser-112 and Ser-155 is known to inhibit its proapoptotic activity. Preincubation with H89 blocked dDAVP-induced phosphorylation of both Bad and Bok, suggesting dependence on protein kinase A (PKA). This study provides evidence that AVP can inhibit apoptosis through the V2 receptor and downstream cAMP-mediated pathways in mammalian kidney. The antiapoptotic action of AVP may be relevant to a number of physiological and pathophysiological conditions including osmotic tolerance in the inner medulla, escape from AVP-induced antidiuresis, and polycystic kidney disease.

Aquaporin-2 regulation in health and disease.

Aquaporin-2 (AQP2), the vasopressin-regulated water channel of the renal collecting duct, is dysregulated in numerous disorders of water balance in people and animals, including those associated with polyuria (urinary tract obstruction, hypokalemia, inflammation, and lithium toxicity) and with dilutional hyponatremia (syndrome of inappropriate antidiuresis, congestive heart failure, cirrhosis). Normal regulation of AQP2 by vasopressin involves 2 independent regulatory mechanisms: (1) short-term regulation of AQP2 trafficking to and from the apical plasma membrane, and (2) long-term regulation of the total abundance of the AQP2 protein in the cells. Most disorders of water balance are the result of dysregulation of processes that regulate the total abundance of AQP2 in collecting duct cells. In general, the level of AQP2 in a collecting duct cell is determined by a balance between production via translation of AQP2 mRNA and removal via degradation or secretion into the urine in exosomes. AQP2 abundance increases in response to vasopressin chiefly due to increased translation subsequent to increases in AQP2 mRNA. Vasopressin-mediated regulation of AQP2 gene transcription is poorly understood, although several transcription factor-binding elements in the 5' flanking region of the AQP2 gene have been identified, and candidate transcription factors corresponding to these elements have been discovered in proteomics studies. Here, we review progress in this area and discuss elements of vasopressin signaling in the collecting duct that may impinge on regulation of AQP2 in health and in the context of examples of polyuric diseases.

Conditional Allele Mouse Planner (CAMP): software to facilitate the planning and design of breeding strategies involving mice with conditional alleles.

Transgenic and conditional knockout mouse models play an important role in biomedical research and their use has grown exponentially in the last 5-10 years. Generating conditional knockouts often requires breeding multiple alleles onto the background of a single mouse or group of mice. Breeding these mice depends on parental genotype, litter size, transmission frequency, and the number of breeding rounds. Therefore, a well planned breeding strategy is critical for keeping costs to a minimum. However, designing a viable breeding strategy can be challenging. With so many different variables this would be an ideal task for a computer program. To facilitate this process, we created a Java-based program called Conditional Allele Mouse Planner (CAMP). CAMP is designed to provide an estimate of the number of breeders, amount of time, and costs associated with generating mice of a particular genotype. We provide a description of CAMP, how to use it, and offer it freely as an application.

Transgenic mice: beyond the knockout.

Transgenic mice have had a tremendous impact on biomedical research. Most researchers are familiar with transgenic mice that carry Cre recombinase (Cre) and how they are used to create conditional knockouts. However, some researchers are less familiar with many of the other types of transgenic mice and their applications. For example, transgenic mice can be used to study biochemical and molecular pathways in primary cultures and cell suspensions derived from transgenic mice, cell-cell interactions using multiple fluorescent proteins in the same mouse, and the cell cycle in real time and in the whole animal, and they can be used to perform deep tissue imaging in the whole animal, follow cell lineage during development and disease, and isolate large quantities of a pure cell type directly from organs. These novel transgenic mice and their applications provide the means for studying of molecular and biochemical events in the whole animal that was previously limited to cell cultures. In conclusion, transgenic mice are not just for generating knockouts.

The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice.

Regulation of sodium balance is a critical factor in the maintenance of euvolemia, and dysregulation of renal sodium excretion results in disorders of altered intravascular volume, such as hypertension. The amiloride-sensitive epithelial sodium channel (ENaC) is thought to be the only mechanism for sodium transport in the cortical collecting duct (CCD) of the kidney. However, it has been found that much of the sodium absorption in the CCD is actually amiloride insensitive and sensitive to thiazide diuretics, which also block the Na-Cl cotransporter (NCC) located in the distal convoluted tubule. In this study, we have demonstrated the presence of electroneutral, amiloride-resistant, thiazide-sensitive, transepithelial NaCl absorption in mouse CCDs, which persists even with genetic disruption of ENaC. Furthermore, hydrochlorothiazide (HCTZ) increased excretion of Na+ and Cl- in mice devoid of the thiazide target NCC, suggesting that an additional mechanism might account for this effect. Studies on isolated CCDs suggested that the parallel action of the Na+-driven Cl-/HCO3- exchanger (NDCBE/SLC4A8) and the Na+-independent Cl-/HCO3- exchanger (pendrin/SLC26A4) accounted for the electroneutral thiazide-sensitive sodium transport. Furthermore, genetic ablation of SLC4A8 abolished thiazide-sensitive NaCl transport in the CCD. These studies establish what we believe to be a novel role for NDCBE in mediating substantial Na+ reabsorption in the CCD and suggest a role for this transporter in the regulation of fluid homeostasis in mice.

P2Y(2) receptors and water transport in the kidney.

The kidneys play a critical role in the maintenance of water homeostasis. This is achieved by the inherent architecture of the nephron along with the expression of various membrane transporters and channels that are responsible for the vectorial transport of salt and water. The collecting duct has become a focus of attention by virtue of its ability to transport water independent of solutes (free-water transport), and its apparent involvement in various water balance disorders. It was originally believed that the water transport capability of the collecting duct was solely under the influence of the circulating hormone, arginine vasopressin (AVP). However, during the past decade, locally produced autocrine and/or paracrine factors have emerged as potent modulators of transport of water by the collecting duct. Recently, much attention has been focused on the purinergic regulation of renal water transport. This review focuses on the role of the P2Y(2) receptor, the predominant purinergic receptor expressed in the collecting duct, in the modulation of water transport in physiological and pathophysiological conditions, and its therapeutic potential as a drug target to treat water balance disorders in the clinic. Studies carried out by us and other investigators are unravelling potent interactions among AVP, prostanoid and purinergic systems in the medullary collecting duct, and the perturbations of these interactions in water balance disorders such as acquired nephrogenic diabetes insipidus. Future studies should address the potential therapeutic benefits of modulators of P2Y(2) receptor signalling in water balance disorders, which are extremely prevalent in hospitalised patients irrespective of the underlying pathology.

Systems-level analysis of cell-specific AQP2 gene expression in renal collecting duct.

We used a systems biology-based approach to investigate the basis of cell-specific expression of the water channel aquaporin-2 (AQP2) in the renal collecting duct. Computational analysis of the 5'-flanking region of the AQP2 gene (Genomatix) revealed 2 conserved clusters of putative transcriptional regulator (TR) binding elements (BEs) centered at -513 bp (corresponding to the SF1, NFAT, and FKHD TR families) and -224 bp (corresponding to the AP2, SRF, CREB, GATA, and HOX TR families). Three other conserved motifs corresponded to the ETS, EBOX, and RXR TR families. To identify TRs that potentially bind to these BEs, we carried out mRNA profiling (Affymetrix) in mouse mpkCCDc14 collecting duct cells, revealing expression of 25 TRs that are also expressed in native inner medullary collecting duct. One showed a significant positive correlation with AQP2 mRNA abundance among mpkCCD subclones (Ets1), and 2 showed a significant negative correlation (Elf1 and an orphan nuclear receptor Nr1h2). Transcriptomic profiling in native proximal tubules (PT), medullary thick ascending limbs (MTAL), and IMCDs from kidney identified 14 TRs (including Ets1 and HoxD3) expressed in the IMCD but not PT or MTAL (candidate AQP2 enhancer roles), and 5 TRs (including HoxA5, HoxA9 and HoxA10) expressed in PT and MTAL but not in IMCD (candidate AQP2 repressor roles). In luciferase reporter assays, overexpression of 3 ETS family TRs transactivated the mouse proximal AQP2 promoter. The results implicate ETS family TRs in cell-specific expression of AQP2 and point to HOX, RXR, CREB and GATA family TRs as playing likely additional roles.

Inactivation of Pkd1 in principal cells causes a more severe cystic kidney disease than in intercalated cells.

Renal cysts in autosomal dominant polycystic kidney disease arise from cells throughout the nephron, but there is an uncertainty as to whether both the intercalated cells (ICs) and principal cells (PCs) within the collecting duct give rise to cysts. To determine this, we crossed mice containing loxP sites within introns 1 and 4 of the Pkd1 gene with transgenic mice expressing Cre recombinase under control of the aquaporin-2 promoter or the B1 subunit of the proton ATPase promoter, thereby generating PC- or IC-specific knockout of Pkd1, respectively. Mice, that had Pkd1 deleted in the PCs, developed progressive cystic kidney disease evident during the first postnatal week and had an average lifespan of 8.2 weeks. There was no change in the cellular cAMP content or membrane aquaporin-2 expression in their kidneys. Cysts were present in the cortex and outer medulla but were absent in the papilla. Mice in which PKd1 was knocked out in the ICs had a very mild cystic phenotype as late as 13 weeks of age, limited to 1-2 cysts and confined to the outer rim of the kidney cortex. These mice lived to at least 1.5 years of age without evidence of early mortality. Our findings suggest that PCs are more important than ICs for cyst formation in polycystic kidney disease.

The V-ATPase B1-subunit promoter drives expression of Cre recombinase in intercalated cells of the kidney.

The collecting duct of the kidney is composed of two morphologically and physiologically distinct cell types, principal and intercalated cells. To better understand intercalated cell function we generated a transgenic mouse expressing Cre recombinase under the control of a cell type- specific promoter. We used 7 kb of the ATP6V1B1 5' untranslated region (B1 promoter), a gene found in the intercalated cells of the kidney and the male reproductive tract. We first crossed these B1-Cre transgenic mice with the ROSA26-loxP-stop-loxP-yellow fluorescent protein reporter mice to assess the specificity of Cre expression. Immunohistochemistry and confocal fluorescence microscopy showed that Cre is selectively active in all intercalated cells (type A, type B, and non-A/B cells) within the collecting duct and most cells of the connecting segment. About half of the principal cells of the connecting segment also expressed Cre, a pattern also seen in B1-driven enhanced green fluorescent protein transgenic mice. Cre was found to be active in the male reproductive tract and at a low level in limited non-ATP6V1B1 expressing tissues. The B1-Cre transgenic mice are healthy, breed normally, produce regular sized litters, and transmit the transgene in Mendelian fashion. This new cell-specific Cre expressing mouse should prove useful for the study of intercalated cell physiology and development.

A small subpopulation of blastospores in candida albicans biofilms exhibit resistance to amphotericin B associated with differential regulation of ergosterol and beta-1,6-glucan pathway genes.

The resistance of Candida albicans biofilms to a broad spectrum of antimicrobial agents has been well documented. Biofilms are known to be heterogeneous, consisting of microenvironments that may induce formation of resistant subpopulations. In this study we characterized one such subpopulation. C. albicans biofilms were cultured in a tubular flow cell (TF) for 36 h. The relatively large shear forces imposed by draining the TF removed most of the biofilm, which consisted of a tangled mass of filamentous forms with associated clusters of yeast forms. This portion of the biofilm exhibited the classic architecture and morphological heterogeneity of a C. albicans biofilm and was only slightly more resistant than either exponential- or stationary-phase planktonic cells. A submonolayer fraction of blastospores that remained on the substratum was resistant to 10 times the amphotericin B dose that eliminated the activity of the planktonic populations. A comparison between planktonic and biofilm populations of transcript abundance for genes coding for enzymes in the ergosterol (ERG1, -3, -5, -6, -9, -11, and -25) and beta-1,6-glucan (SKN and KRE1, -5, -6, and -9) pathways was performed by quantitative RT-PCR. The results indicate a possible association between the high level of resistance exhibited by the blastospore subpopulation and differential regulation of ERG1, ERG25, SKN1, and KRE1. We hypothesize that the resistance originates from a synergistic effect involving changes in both the cell membrane and the cell wall.

Automated method for the isolation of collecting ducts.

The structural and functional heterogeneity of the collecting duct present a tremendous experimental challenge requiring manual microdissection, which is time-consuming, labor intensive, and not amenable to high throughput. To overcome these limitations, we developed a novel approach combining the use of transgenic mice expressing green fluorescent protein (GFP) in the collecting duct with large-particle-based flow cytometry to isolate pure populations of tubular fragments from the whole collecting duct (CD), or inner medullary (IMCD), outer medullary (OMCD), or connecting segment/cortical collecting duct (CNT/CCD). Kidneys were enzymatically dispersed into tubular fragments and sorted based on tubular length and GFP intensity using large-particle-based flow cytometry or a complex object parametric analyzer and sorter (COPAS). A LIVE/DEAD assay demonstrates that the tubules were >90% viable. Tubules were collected as a function of fluorescent intensity and analyzed by epifluorescence and phase microscopy for count accuracy, GFP positivity, average tubule length, and time required to collect 100 tubules. Similarly, mRNA and protein from sorted tubules were analyzed for expression of tubule segment-specific genes using quantitative real-time RT-PCR and immunoblotting. The purity and yield of sorted tubules were related to sort stringency. Four to six replicates of 100 collecting ducts (9.68+/-0.44-14.5+/-0.66 cm or 9.2+/-0.7 mg tubular protein) were routinely obtained from a single mouse in under 1 h. In conclusion, large-particle-based flow cytometry is fast, reproducible, and generates sufficient amounts of highly pure and viable collecting ducts from single or replicate animals for gene expression and proteomic analysis.

Expression and function of COX isoforms in renal medulla: evidence for regulation of salt sensitivity and blood pressure.

Expression of cyclooxygenase (COX)-2, but not COX-1, in the renal medulla is stimulated by chronic salt loading; yet the functional implication of this phenomenon is incompletely understood. The present study examined the cellular localization and antihypertensive function of high-salt-induced COX-2 expression in the renal medulla, with a parallel assessment of the function of COX-1. COX-2 protein expression in response to high-salt loading, assessed by immunostaining, was found predominantly in inner medullary interstitial cells, whereas COX-1 protein was abundant in collecting duct (CD) and inner medullary interstitial cells and was not affected by high salt. We compared mRNA expressions of COX-1 and COX-2 in CD vs. non-CD cells isolated from aquaporin 2-green fluorescent protein transgenic mice. A low level of COX-2 mRNA, but a high level of COX-1 mRNA, as determined by real-time RT-PCR, was detected in CD compared with non-CD segments. During high-salt intake, chronic infusions of the COX-2 blocker NS-398 and the COX-1 blocker SC-560 into the renal medulla of Sprague-Dawley rats for 5 days induced approximately 30- and 15-mmHg increases in mean arterial pressure, respectively. During similar high-salt intake, COX-1 knockout mice exhibited a gradual, but significant, increase in systolic blood pressure that was associated with a marked suppression of urinary PGE2 excretion. Therefore, we conclude that the two COX isoforms in the renal medulla play a similar role in the stabilization of arterial blood pressure during salt loading.

Chronic dDAVP infusion in rats decreases the expression of P2Y2 receptor in inner medulla and P2Y2 receptor-mediated PGE2 release by IMCD.

Activation of P2Y2 receptor (P2Y2-R) in inner medullary collecting duct (IMCD) of rat decreases AVP-induced water flow and releases PGE(2). We observed that dehydration of rats decreases the expression of P2Y2 receptor in inner medulla (IM) and P2Y2-R-mediated PGE(2) release by IMCD. Because circulating vasopressin (AVP) levels are increased in dehydrated condition, we examined whether chronic infusion of desmopressin (dDAVP) has a similar effect on the expression and activity of P2Y2-R. Groups of rats were infused with saline or dDAVP (5 or 20 ng/h sc, 5 or 6 days) via osmotic minipumps and euthanized. Urine volume, osmolality, and PGE(2) metabolite content were determined. AQP2- and P2Y2- and V2-R mRNA and/or protein in IM were quantified by real-time RT-PCR and immunoblotting, respectively. P2Y2-R-mediated PGE(2) release by freshly prepared IMCD was assayed using ATPgammaS as a ligand. Chronic dDAVP infusion resulted in low-output of concentrated urine and significantly increased the AQP2 protein abundance in IM. On the contrary, dDAVP infusion at 5 or 20 ng/h significantly decreased P2Y2-R protein abundance (approximately 40% of saline-treated group). In parallel, the relative expression of P2Y2-R vs. AQP2- or V2-R mRNA was significantly decreased. Furthermore, the P2Y2-R-mediated PGE(2) release by IMCD was significantly decreased in rats infused 20 ng/h but not 5 ng/h of dDAVP. Urinary PGE(2) metabolite excretion, however, did not change with dDAVP infusion. In conclusion, chronic dDAVP infusion decreases the expression and activity of P2Y2-R in IM. This may be due to a direct effect of dDAVP or dDAVP-induced increase in medullary tonicity.

P2Y2 receptor mRNA and protein expression is altered in inner medullas of hydrated and dehydrated rats: relevance to AVP-independent regulation of IMCD function.

Arginine vasopressin (AVP), acting through a cAMP second messenger system, regulates osmotic water permeability (Pf) of the collecting duct. In the collecting duct, the activities of cAMP and phosphonositides (PI) are mutually inhibitory. The P2Y2 receptor (P2Y2-R) is a G protein-coupled extracellular nucleotide receptor associated with PI signaling pathway. Previously, we showed that P2Y2-R is expressed in inner medullary collecting duct (IMCD) of rat, and its agonist (ATP/UTP) activation decreased AVP-induced Pf and resulted in enhanced production of prostaglandin E2. Hydrated and dehydrated states are associated with alterations in the circulating levels of AVP, expression and/or subcellular distribution of AVP-regulated aquaporin-2 water channel in IMCD and thus Pf of IMCD. We hypothesized that altered expression and/or signaling via P2Y2-R may also modulate IMCD function in these conditions. Sprague-Dawley rats were subjected to dehydration by water deprivation (48 h) or hydration (48 or 96 h) by providing sucrose water. Hydration or dehydration resulted in marked alterations in mRNA expression (Northern blot analysis and real-time RT-PCR) and protein abundance (Western blot analysis) of P2Y2-R, with hydrated rats showing significantly higher levels compared with dehydrated rats. Sequential hydration and dehydration experiments also revealed that the regulated expression profiles of P2Y2-R mRNA and protein are discordant. Conversely, the expression of V2-R mRNA remained unaltered during hydration and dehydration. Because virtually all renal cells release ATP in a regulated fashion, the observed alterations in P2Y2-R expression in the inner medulla in hydrated and dehydrated states may constitute a novel mechanism of purinergic modulation of IMCD function.

V-ATPase B1-subunit promoter drives expression of EGFP in intercalated cells of kidney, clear cells of epididymis and airway cells of lung in transgenic mice.

The kidney, epididymis, and lungs are complex organs with considerable epithelial cell heterogeneity. This has limited the characterization of pathophysiological transport processes that are specific for each cell type in these epithelia. The purpose of the present study was to develop new tools to study cell-specific gene and protein expression in such complex tissues and organs. We report the production of a transgenic mouse that expresses enhanced green fluorescent protein (EGFP) in a subset of epithelial cells that express the B1 subunit of vacuolar H(+)-ATPase (V-ATPase) and are actively involved in proton transport. A 6.5-kb portion of the V-ATPase B1 promoter was used to drive expression of EGFP. In two founders, quantitative real-time RT-PCR demonstrated expression of EGFP in kidney, epididymis, and lung. Immunofluorescence labeling using antibodies against the B1 and E subunits of V-ATPase and against carbonic anhydrase type II (CAII) revealed specific EGFP expression in all renal type A and type B intercalated cells, some renal connecting tubule cells, all epididymal narrow and clear cells, and some nonciliated airway epithelial cells. No EGFP expression was detected in collecting duct principal cells (identified using an anti-AQP2 antibody) or epididymal principal cells (negative for V-ATPase or CAII). This EGFP-expressing mouse model should prove useful in future studies of gene and protein expression and their physiological and/or developmental regulation in distinct cell types that can now be separated using fluorescence-assisted microdissection, fluorescence-activated cell sorting, and laser capture microdissection.