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Sphingosine kinase 1 (SphK1) and SphK2 are ubiquitous enzymes that generate sphingosine-1-phosphate (S1P), a ligand for a family of G protein-coupled receptors (S1PR1-S1PR5) with important functions in the vascular and immune systems. Here we explore the role of these kinases and receptors in recovery from anaphylaxis in mice. We found that [Sphk2.sup.[-/-]] mice had a rapid recovery from anaphylaxis. In contrast, [Sphk1.sup.[-/-]] mice showed poor recovery from anaphylaxis and delayed histamine clearance. Injection of S1P into [Sphk1.sup.[-/-]] mice increased histamine clearance and promoted recovery from anaphylaxis. Adoptive cell transfer experiments demonstrated that SphK1 activity was required in both the hematopoietic and nonhematopoietic compartments for recovery from anaphylaxis. Mice lacking the S1P receptor S1PR2 also showed a delay in plasma histamine clearance and a poor recovery from anaphylaxis. However, S1P did not promote the recovery of [S1pr2.sup.[-/-]] mice from anaphylaxis, whereas [S1pr2.sup.±] mice showed partial recovery. Unlike [Sphk2.sup.[-/-]] mice, [Sphk1.sup.[-/-]] and [S1pr2.sup.[-/-]] mice had severe hypotension during anaphylaxis. Thus, SphK1-produced S1P regulates blood pressure, histamine clearance, and recovery from anaphylaxis in a manner that involves S1PR2. This suggests that specific S1PR2 agonists may serve to counteract the vasodilation associated with anaphylactic shock., Introduction Anaphylaxis is a serious allergic reaction that typically involves the activation of mast cells and basophils (1-3). Unlike other allergic reactions involving mast cells, anaphylaxis affects the function of [...]

Levels of the necessary nutrient vitamin C (ascorbate) are tightly regulated by intestinal absorption, tissue accumulation, and renal reabsorption and excretion. Ascorbate levels are controlled in part by regulation of transport through at least 2 sodium-dependent transporters: Slc23a1 and Slc23a2 (also known as Svct1 and Svct2, respectively). Previous work indicates that Slc23a2 is essential for viability in mice, but the roles of Slc23a1 for viability and in adult physiology have not been determined. To investigate the contributions of Slc23a1 to plasma and tissue ascorbate concentrations in vivo, we generated [Slc23a1.sup.[-/-]] mice. Compared with wild-type mice, [Slc23a1.sup.[-/-]] mice increased ascorbate fractional excretion up to 18-fold. Hepatic portal ascorbate accumulation was nearly abolished, whereas intestinal absorption was marginally affected. Both heterozygous and knockout pups born to [Slc23a1.sup.[-/-]] dams exhibited approximately 45% perinatal mortality, and this was associated with lower plasma ascorbate concentrations in dams and pups. Perinatal mortality of [Slc23a1.sup.[-/-]] pups born to [Slc23a1.sup.[-/-]] dams was prevented by ascorbate supplementation during pregnancy. Taken together, these data indicate that ascorbate provided by the dam influenced perinatal survival. Although [Slc23a1.sup.[-/-]] mice lost as much as 70% of their ascorbate body stores in urine daily, we observed an unanticipated compensatory increase in ascorbate synthesis. These findings indicate a key role for Slc23a1 in renal ascorbate absorption and perinatal survival and reveal regulation of vitamin C biosynthesis in mice., Introduction Vitamin C (ascorbate) is synthesized by most mammals. Humans lack the terminal enzyme gulonolactone oxidase in the synthesis pathway and rely on dietary intake for ascorbate (1). Ascorbate is [...]

Adenosine 1 receptors (A1AR) in the kidney are expressed in the vasculature and the tubular system. Pharmacological inhibition or global genetic deletion of A1AR causes marked reductions or abolishment of tubuloglomerular feedback (TGF) responses. To assess the function of vascular A1AR in TGF, we generated transgenic mouse lines in which A1AR expression in smooth muscle was augmented by placing A1AR under the control of a 5.38-kb fragment of the rat smooth muscle [alpha]-actin promoter and first intron (12). Two founder lines with highest expression in the kidney [353 [+ or -] 42 and 575 [+ or -] 43% compared with the wild type (WT)] were used in the experiments. Enhanced expression of A1AR at the expected site in these lines was confirmed by augmented constrictor responses of isolated afferent arterioles to administration of the A1AR agonist [N.sup.6]-cyclohexyladenosine. Maximum TGF responses (0-30 nl/min flow step) were increased from 8.4 [+ or -] 0.9 mmHg in WT (n = 21) to 14.2 [+ or -] 0.7 mmHg in A1AR-transgene (tg) 4 (n = 22; P < 0.0001), and to 12.6 [+ or -] 1.2 mmHg in A1AR-tg7 (n = 12; P < 0.02). Stepwise changes in perfusion flow caused greater numerical TGF responses in A1AR-tg than WT in all flow ranges with differences reaching levels of significance in the intermediate flow ranges of 7.5-10 and 10-15 nl/min. Proximal-distal single-nephron glomerular filtration rate (SNGFR) differences (free-flow micropuncture) were also increased in A1AR-tg, averaging 6.25 [+ or -] 1.5 nl/min compared with 2.6 [+ or -] 0.51 nl/min in WT (P = 0.034). Basal plasma renin concentrations as well as the suppression of renin secretion after volume expansion were similar in A1AR-tg and WT mice, suggesting lack of transgene expression in juxtaglomerular cells. These data indicate that A1AR expression in vascular smooth muscle cells is a critical component for TGF signaling and that changes in renal vascular A1AR expression may determine the magnitude of TGF responses. single nephron glomerular filtration rate; smooth muscle; juxtaglomerular apparatus doi: 10.1152/ajprenal.00264.2009.

X-linked nephrogenic diabetes insipidus (XNDI) is a severe kidney disease caused by inactivating mutations in the V2 vasopressin receptor (V2R) gene that result in the loss of renal urine-concentrating ability. At present, no specific pharmacological therapy has been developed for XNDI, primarily due to the lack of suitable animal models. To develop what we believe to be the first viable animal model of XNDI, we generated mice in which the V2R gene could be conditionally deleted during adulthood by administration of 4-OH-tamoxifen. Radioligand-binding studies confirmed the lack of V2R-binding sites in kidneys following 4-OH-tamoxifen treatment, and further analysis indicated that upon V2R deletion, adult mice displayed all characteristic symptoms of XNDI, including polyuria, polydipsia, and resistance to the antidiuretic actions of vasopressin. Gene expression analysis suggested that activation of renal EP4 [PGE.sub.2] receptors might compensate for the lack of renal V2R activity in XNDI mice. Strikingly, both acute and chronic treatment of the mutant mice with a selective EP4 receptor agonist greatly reduced all major manifestations of XNDI, including changes in renal morphology. These physiological improvements were most likely due to a direct action on EP4 receptors expressed on collecting duct cells. These findings illustrate the usefulness of the newly generated V2R mutant mice for elucidating and testing new strategies for the potential treatment of humans with XNDI., Introduction In nephrogenic diabetes insipidus (NDI), the kidney is unable to conserve water despite normal or increased plasma levels of the antidiuretic hormone arginine vasopressin (AVP) (1-5). Most patients (> [...]

During metanephric kidney development, renin expression in the renal vasculature begins in larger vessels, shifting to smaller vessels and finally remaining restricted to the terminal portions of afferent arterioles at the entrance into the glomerular capillary network. The mechanisms determining the successive expression of renin along the vascular axis of the kidney are not well understood. Since the cAMP signaling cascade plays a central role in the regulation of both renin secretion and synthesis in the adult kidney, it seemed feasible that this pathway might also be critical for renin expression during kidney development. In the present study we determined the spatiotemporal development of renin expression and the development of the preglomerular arterial tree in mouse kidneys with renin cell-specific deletion of [G.sub.s][alpha], a core element for receptor activation of adenylyl cyclases. We found that in the absence of the [G.sub.s][alpha] protein, renin expression was largely absent in the kidneys at any developmental stage, accompanied by alterations in the development of the preglomerular arterial tree. These data indicate that the maintenance of renin expression following a specific spatiotemporal pattern along the preglomerular vasculature critically depends on the availability of [G.sub.s][alpha]. We infer from our data that the cAMP signaling pathway is not only critical for the regulation of renin synthesis and secretion in the mature kidney but that it also is critical for establishing the juxtaglomerular expression site of renin during development. fetal kidney; transgenic mice

The mechanisms underlying the development and prevention of diabetic nephropathy are still not fully understood. In the present study in the Otsuka Long-Evans Tokushima Fatty (OLETF) model of type 2 diabetic rats, we investigated whether renal hemodynamic abnormalities exist and whether they precede the onset of diabetes. Using OLETF rats in both prediabetic and diabetic stages, we assessed autoregulatory responses of total renal blood flow (RBF) and of superficial (SBF) and deep renal cortical (DBF) blood flow to stepwise reductions of renal perfusion pressure (RPP) induced by a manual clamp on the abdominal aorta. During clamp-induced reductions of RPP by 10 or 20 mmHg, RBF fell significantly more in OLETF rats than in lean control [Long-Evans Tokushima Otsuka (LETO)] rats. Whereas SBF showed no significant changes in either OLETF rats or LETO rats during mild clamping, DBF decreased significantly more in OLETF rats than LETO rats. Reduced autoregulatory efficiency in OLETF rats was observed in both prediabetic and diabetic stages. Micropuncture studies showed that tubuloglomerular feedback (TGF) responses of stop flow pressure are reduced in prediabetic (-7.3 vs. -25.7%) as well as in diabetic OLETF rats compared with LETO control rats (-4.4 vs. -18.8%). Renal corticotomy was performed to measure glomerular capillary pressure ([P.sub.gc]) directly. [P.sub.gc] of deep cortical glomeruli was higher than superficial glomerular [P.sub.gc] in both strains of rats, but the difference was especially pronounced in OLETF rats (deep 78 [+ or -] 2 vs. superficial 57 [+ or -] 4 mmHg). This study demonstrates reduced autoregulatory adjustments and impaired TGF efficiency in prediabetic OLETF rats. Thus abnormal RBF regulation precedes the onset of diabetes and is especially pronounced in the deep cortical region. type 2 diabetes; hemodynamics; deep nephrons; corticotomy; micropuncture; Otsuka Long-Evans Tokushima Fatty

IA-2 and IA-2[beta], major autoantigens in type 1 diabetes, are transmembrane proteins in dense-core vesicles, and their expression influences the secretion of hormones and neurotransmitters. The present experiments were performed to examine whether IA-2 and IA-2[beta] modulate the release of renin from dense-core vesicles of juxtaglomerular granular cells in the kidney. Plasma renin concentration (PRC; ng angiotensin I * [ml.sup.-1] * [h.sup.-1]) was significantly reduced in mice with null mutations in IA-2, IA-2[beta], or both IA-2 and IA-2[beta] compared with wild-type mice (876 [+ or -] 113, 962 [+ or -] 130, and 596 [+ or -] 82 vs. 1,367 + 93; P < 0.01, P < 0.02, and P < 0.001). Renin mRNA levels were reduced to 26.4 [+ or -] 5.1, 39 [+ or -] 5.4, and 35.3 [+ or -] 5.5% of wild-type in IA-2-/-, IA-2[beta]-/-, and IA-2/IA-2[beta]-/- mice. Plasma aldosterone levels were not significantly different among genotypes. The regulation of PRC by furosemide and salt intake, and of aldosterone by salt intake, was maintained in all genotypes. IA-2 and IA-2[beta] expression did not colocalize with renin but showed overlapping immunoreactivity with tyrosine hydroxylase. While propranolol reduced PRC in wild-type mice, it had no effect on PRC in IA-2/ IA-2[beta]-/- mice. Renal tyrosine hydroxylase mRNA and immunoreactivity were reduced in IA-2/IA-2[beta]-/- mice as was the urinary excretion of catecholamines. We conclude that IA-2 and IA-2[beta] are required to maintain normal levels of renin expression and renin release, most likely by permitting normal rates of catecholamine release from sympathetic nerve terminals. knockout mice; renin mRNA; propranolol; blood pressure; heart rate; salt intake

NKCC1 is a widely expressed isoform of the Na-2Cl-K cotransporter that mediates several direct and indirect vascular effects and regulates expression and release of renin. In this study, we used NKCC1-deficient ([NKCC1.sup.-/-]) and wild-type (WT) mice to assess day/night differences of blood pressure (BP), locomotor activity, and renin release and to study the effects of high (8%) or low (0.03%) dietary NaCl intake on BP, activity, and the renin/aldosterone system. On a standard diet, 24-h mean arterial blood pressure (MAP) and heart rate determined by radiotelemetry, and their day/night differences, were not different in [NKCC1.sup.-/-] and WT mice. Spontaneous and wheel-running activities in the active night phase were lower in [NKCC1.sup.-/-] than WT mice. In [NKCC1.sup.-/-] mice on a high-NaCl diet, MAP increased by 10 mmHg in the night without changes in heart rate. In contrast, there was no salt-dependent blood pressure change in WT mice. MAP reductions by hydralazine (1 mg/kg) or isoproterenol (10 [micro]g/mouse) were significantly greater in [NKCC1.sup.-/-] than WT mice. Plasma renin (PRC; ng ANG I x [ml.sup.-1] x [h.sup.i]) and aldosterone (aldo; pg/ml) concentrations were higher in [NKCC1.sup.-/-] than WT mice (PRC: 3,745 [+ or -] 377 vs. 1,245 [+ or -] 364; aldo: 763 [+ or -] 136 vs. 327 [+ or -] 98). Hyperreninism and hyperaldosteronism were found in [NKCC1.sup.-/-] mice during both day and night. High Na suppressed PRC and aldosterone in both [NKCC1.sup.-/-] and WT mice, whereas a low-Na diet increased PRC and aldosterone in WT but not [NKCC1.sup.-/-] mice. We conclude that 24-h MAP and MAP circadian rhythms do not differ between [NKCCI.sup.-/-] and WT mice on a standard diet, probably reflecting a balance between anti- and prohypertensive factors, but that blood pressure of [NKCC1.sup.-/-] mice is more sensitive to increases and decreases of Na intake. radiotelemetry; running wheels; renin; aldosterone; vasodilators; sodium-2 chloride-potassium cotransporter-deficient mice

The renal Na-K-2Cl cotransporter (NKCC2, BSCI) is selectively expressed in the apical membrane of cells of the thick ascending limb of the loop of Henle (TAL) and macula densa. NKCC2-dependent salt transport constitutes the major apical entry pathway for transepithelial salt reabsorption in the TAL. Although NKCC2 is encoded by a single gene (Slcl2al), differential splicing of the NKCC2 pre-mRNA results in the formation of several alternate transcripts. Thus three full-length splice isoforms of NKCC2 differ in their variable exon 4, resulting in transcripts for NKCC2B, NKCC2A, and NKCC2F. In addition to full-length isoforms, variants with truncated COOH-terminal ends have been described. The various splice isoforms of NKCC2 differ in their localization along the TAL and in their transport characteristics. Data in the literature are reviewed to assess the principles of NKCC2 differential splicing, the localization of NKCC2 splice isoforms along the TAL in various species, and the functional characteristics of the splice isoforms. In addition, we discuss the functional significance of NKCC2 isoforms for TAL salt retrieval and for the specific salt sensor function of macula densa cells based on studies using isoform-specific NKCC2-knockout mice. We suggest that different NKCC2 splice variants cooperate in salt retrieval 'along the TAL and that the coexpression of two splice variants (NKCC2B and NKCC2A) in the macula densa cells facilitates efficient salt sensing over wide ranges of fluctuating salt concentrations. differential splicing; macula densa; tubuloglomerular feedback; renin

The [beta]-adrenergic pathway has been considered one important effector of circadian variation in arterial pressure. Experiments were performed in [beta]1/[beta]2-adrenergic receptor-deficient mice ([beta]1[beta]2ADR-/-) to assess whether this pathway is required for circadian variation in mean arterial pressure (MAP) and to determine the impact of its loss on the response to changes in dietary salt. Twenty-four-hour recordings of MAP, heart rate (HR), and locomotor activity were made in conscious 16- to 17-wk-old mice [wild-type, (WT), n = 7; [beta]1/[beta]2ADR-/-, n = 10] by telemetry. Both WT and [beta]1/[beta]2ADR-/- mice demonstrated robust circadian variation in MAP and HR, although 24-h mean MAP was 10% lower (102.02 [+ or -] 1.81 vs. 92.11 [+ or -] 2.62 mmHg) in [beta]/ [beta]2ADR-/- than WT, HR was 16% lower and day-night differences reduced. Both WT and [beta]1/[beta]2ADR-/- mice adapted to changed salt intake without changed MAP. However, the [beta]1/[beta]2ADR-/- mice demonstrated a striking reduction in locomotor activity in light and dark phases of the day. In WT mice, MAP was markedly affected by locomotor activity, resulting in bimodal distributions in both light and dark. When MAP was analyzed using only intervals without locomotor activity, bimodality and circadian differences were reduced, and there was no significant difference between the two genotypes. The results indicate that there is no direct effect or role for the [beta]-adrenergic system in circadian variation of arterial pressure in mice, aside from the indirect consequences of altered locomotor activity. Our results also confirm that locomotor activity contributes strongly to circadian variation in blood pressure in mice. salt intake; sympathetic nervous system

Studies in mice with null mutations of adenosine 1 receptor or ecto-5'-nucleotidase genes suggest a critical role of adenosine and its precursor 5'-AMP in tubulovascular signaling. To assess whether the source of juxtaglomerular nucleotides can be traced back to ATP dephosphorylation, experiments were performed in mice with a deficiency in NTPDasel/ CD39, an ecto-ATPase catalyzing the formation of AMP from ATP and ADP. Urine osmolarity and glomerular filtration rate (GFR) were indistinguishable between NTPDasel/CD[39.sup.-/-] and wild-type (WT) mice. Maximum tubuloglomerular feedback (TGF) responses, as determined by proximal tubular stop flow pressure measurements, were reduced in NTPDasel/CD[39.sup.-/-] mice compared with controls (4.2 [+ or -] 0.9 vs. 10.5 [+ or -] 1.2 mmHg, respectively; P = 0.0002). Residual TGF responses gradually diminished after repeated changes in tubular perfusion flow averaging 2.9 [+ or -] 0.9 (on response) and 3.5 [+ or -] 1.1 (off response) mmHg after the second and 2.2 [+ or -] 0.5 (on response) and 1.5 [+ or -] 0.8 (off response) mmHg after the third challenge, whereas no fading of TGF responsiveness was observed in WT mice. Macula densa-dependent and pressure-dependent inhibition of renin secretion, as assessed by acute salt loading and phenylephrine injection, respectively, were intact in NTPDasel/CD39-deficient mice. In summary, NTPDasel/CD39-deficient mice showed a markedly compromised TGF regulation of GFR. These data support the concept of an extracellular dephosphorylation cascade during tubular-vascular signal transmission in the juxtaglomerular apparatus that is initiated by a regulated release of ATP from macula densa cells and results in adenosine-mediated afferent arteriole constriction. adenosine 5'-triphosphate; CD73; stop flow pressure; phenylephrine; adenosine

Activation of adenosine [A.sub.1] receptors ([A.sub.1]R) can inhibit arginine vasopressin (AVP)-induced cAMP formation in isolated cortical and medullary collecting ducts. To assess the in vivo consequences of the absence of [A.sub.1]R, we performed experiments in mice lacking [A.sub.1]R ([A.sub.1][R.sup.-/-]). We assessed the effects of the vasopressin [V.sub.2] receptor ([V.sub.2]R) agonist 1-desamino-8-D-arginine vasopressin (dDAVP) on cAMP formation in isolated inner medullary collecting ducts (IMCD) and on water excretion in conscious water-loaded mice. dDAVP-induced cAMP formation in isolated IMCD was significantly greater (~2-fold) in [A.sub.1][R.sup.-/-] compared with wild-type mice (WT) and, in contrast to WT, was not inhibited by the [A.sub.1]R agonist N6-cyclohexyladenosine. [A.sub.1][R.sup.-/-] and WT had similar basal urinary excretion of vasopressin, expression of aquaporin-2 protein in renal cortex and medulla, and acute increases in urinary flow rate and electrolyte-free water clearance in response to the [V.sub.2]R antagonist SR121463 or acute water loading; the latter increased inner medullary [A.sub.1]R expression in WT. Dose dependence of dDAVP-induced antidiuresis after acute water loading was not different between the genotypes. However, [A.sub.1][R.sup.-/-] had greater inner medullary expression of cyclooxygenase-1 under basal conditions and of the [P2Y.sub.2] and [EP.sub.3] receptor in response to water loading compared with WT mice. Thus vasopressin-induced cAMP formation is enhanced in isolated IMCD of mice lacking [A.sub.1]R, but the adenosine-[A.sub.1]R/[V.sub.2]R interaction demonstrated in vitro is likely compensated in vivo by multiple mechanisms, a number of which can be 'uncovered' by water loading. arginine vasopressin; aquaporin-2; cyclooxygenase

Author(s): Lane Bekar (corresponding author) [1, 3]; Witold Libionka [1, 3]; Guo-Feng Tian [1]; Qiwu Xu [1]; Arnulfo Torres [1]; Xiaohai Wang [1]; Ditte Lovatt [1]; Erika Williams [1]; Takahiro [...]

Loop diuretics like furosemide have been shown to cause renal vasodilatation in dogs and humans, an effect thought to result from both a direct vascular dilator effect and from inhibition of tubuloglomerular feedback. In isolated peffused afferent arterioles preconstricted with angiotensin II or [N.sup.G]-nitro-L-arginine methyl ester, furosemide caused a dose-dependent increase of vascular diameter, but it was without effect in vessels from NKCC1-/- mice suggesting that inhibition of NKCC1 mediates dilatation in afferent arterioles. In the intact kidney, however, furosemide (2 mg/kg iv) caused a 50.5 [+ or -] 3% reduction of total renal blood flow (RBF) and a 27% reduction of superficial blood flow (SBF) accompanied by a marked and immediate increase of tubular pressure and volume. At 10 mg/kg, furosemide reduced RBF by 60.4 [+ or -] 2%. Similarly, [NKCC1.sup.-/-] mice responded to furosemide with a 45.4% decrease of RBF and a 29% decrease of SBF. Decreases in RBF and SBF and increases of tubular pressure by furosemide were ameliorated by renal decapsulation. In addition, pretreatment with candesartan (2 mg/kg) or indomethacin (5 mg/kg) attenuated the reduction of RBF and peak urine flows caused by furosemide. Our data indicate that furosemide, despite its direct vasodilator potential in isolated afferent arterioles, causes a marked increase in flow resistance of the vascular bed of the intact mouse kidney. We suggest that generation of angiotensin II and/or a vasoconstrictor prostaglandin combined with compression of peritubular capillaries by the expanding tubular compartment are responsible for the reduction of RBF in vivo. renal blood flow; superficial blood flow; candesartan; decapsulation; tubular pressure; NKCC1 knockout doi:10.1152/ajprenal.00073.2007

In the current experiments, we determined the response of plasma renin concentration (PRC) to acute intraperitoneal administration of furosemide (40 mg/kg), hydralazine (2 mg/kg), isoproterenol (10 mg/kg), candesartan (50 [micro]g), or quinaprilate (50 [micro]g) in conscious wild-type (WT) and cyclooxygenase (COX)-2-/- mice on three different genetic backgrounds (mixed, C57BL/6, 129J). PRC was measured in plasma obtained by tail vein puncture. Basal PRC was significantly lower in COX-2-/- than WT mice independent of genetic background (51, 10, and 17% of WT in mixed, 129J, and C57BL/6). All five acute interventions caused significant increases of PRC in both COX-2+/+ and -/- mice, but the response was consistently less in COX-2-deficient mice (e.g., [DELTA]PRC in ng ANG I * [ml.sup.-1] * [h.sup.-1] caused by furosemide, isoproterenol, hydralazine, quinaprilate, or candesartan 4,699 [+ or -] 544, 3,534 [+ or -] 957, 2,522 [+ or -] 369, 9,453 [+ or -] 1,705, 66,455 [+ or -] 21,938 in 129J WT, and 201 [+ or -] 78, 869 [+ or -] 275, 140 [+ or -] 71, 902 [+ or -] 304, 2,660 [+ or -] 954 in 129J COX-2-/-). A low-NaCl diet and enalapril for 1 wk caused a 14-fold elevation of PRC in COX-2-/- mice and was associated with a greatly increased PRC response to acute furosemide ([DELTA]PRC 201 [+ or -] 78 before and 15,984 [+ or -] 2,397 after low Na/enalapril). As measured by radiotelemetry, blood pressure and heart rate responses to furosemide, hydralazine, isoproterenol, candesartan, or quinaprilate were not different between COX-2 genotypes. In conclusion, chronic absence of COX-2 reduces renin expression, release, and PRC and is associated with a reduced ability to alter PRC during acute stimulation regardless of the nature of the stimulus. COX-2 activity does not appear to be a mandatory and specific requirement for furosemide-stimulated renin secretion. furosemide; hydralazine; isoproterenol; quinaprilate; candesartan; genetic background; cyclooxygenase-2

By crossing mice with expression of Cre recombinase under control of the endogenous renin promoter (Sequeira Lopez ML, Pentz ES, Nomasa T, Smithies O, Gomez RA. Dev Cell 6: 719-728, 2004) with mice in which exon 1 of the Gnas gene was flanked by loxP sites (Chen M, Gavrilova O, Liu J, Xie T, Deng C, Nguyen AT, Nackers LM, Lorenzo J, Shen L, Weinstein LS. Proc Natl Acad Sci USA), we generated animals with preferential and nearly complete excision of Gs[alpha] in juxtaglomemlar granular (JG) cells. Compared with wild-type animals, mice with conditional Gs[alpha] deficiency had markedly reduced basal levels of renin expression and very low plasma renin concentrations. Furthermore, the acute release responses to furosemide, hydralazine, and isoproterenol were virtually abolished. Consistent with a state of primary renin depletion, Gs[alpha]-deficient mice had reduced arterial blood pressure, reduced levels of aldosterone, and a low glomemlar filtration rate. Renin content and renin secretion of JG cells in primary culture were drastically reduced, and the stimulatory response to the addition of [PGE.sub.2] or isoproterenol was eliminated. Unexpectedly, Gs[alpha] recombination was also observed in the renal medulla, and this was associated with a vasopressin-resistant concentrating defect. Our study shows that Cre recombinase under control of the renin promoter can be used for the excision of floxed targets from JG cells. We conclude that Gs[alpha]-mediated signal transduction is essential and nonredundant in the control of renin synthesis and release. juxtaglomerular granular cell culture; furosemide; hydralazine; aldosterone; cyclooxygenase 2; urine concentration

Collecting ducts are a major site of renal production and action of both prostaglandins and nitric oxide. Experiments were undertaken to examine whether nitric oxide regulates cyclooxygenase (COX)-2 expression and [PGE.sub.2] release in cultured collecting duct cells. In mIMCD-K2 cells, sodium nitroprusside (SNP) in the 50- to 800-[micro]M range induced a marked dose- and time-dependent increase in COX-2 protein levels, determined by immunoblotting, and the induction was detectable at 4 h. This was preceded by induction of COX-2 mRNA as determined by real-time-RT-PCR. The COX-2 induction was accompanied by a significant rise in [PGE.sub.2] release as determined by enzyme immunoassay. S-nitroso-N-acetylpenicillamine (SNAP) had a similar stimulatory effect on COX-2 expression and [PGE.sub.2] release. 8-bromo-cGMP (200 [micro]M) had no effect on COX-2 expression. The SNP-stimulated COX-2 expression was not affected by the guanylyl cyclase inhibitor methylene blue or the protein kinase G inhibitor KT-5823 (2.0 [micro]M). In contrast, the SNP-stimulated COX-2 expression was significantly reduced by either the Erk1/2 inhibitor PD-98059 or the P38 inhibitor SB-203580 and was abolished by combination of the two kinase inhibitors. The stimulation was also significantly blocked by the SOD mimetic tempol. Thus we conclude that NO stimulates COX-2 expression in collecting duct cells through mechanisms involving MAP kinase and superoxide, but not cGMP. cyclooxygenase-2

To examine ischemic tolerance in the absence of [A.sub.1] adenosine receptors ([A.sub.1]ARs), isolated wild-type (WT) and [A.sub.1]AR knockout ([A.sub.1]KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either [A.sub.1]AR deletion and/or ischemia-reperfusion. [A.sub.1]KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by [A.sub.1]AR deletion. After 20 min of ischemia, CF was attenuated in [A.sub.1]KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A1KO hearts (54.4 [+ or -] 5.1 vs. WT 81.1 [+ or -] 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in [A.sub.1]KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of [A.sub.1]AR transcript in [A.sub.1]KO hearts, and the message for [A.sub.2A], [A.sub.2B] and [A.sub.3] adenosine receptors was similar in uninstrumented [A.sub.1]KO and WT hearts. Ischemia-reperfusion increased [A.sub.2B] mRNA expression 2.5-fold in both WT and [A.sub.1]KO hearts without changing [A.sub.1] or [A.sub.3] expression. In WT hearts, ischemia transiently doubled [A.sub.2A] mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in [A.sub.1]KO hearts. Together, these data affirm the cardioprotective role of [A.sub.1]ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts. gene expression; coronary flow; lactate dehydrogenase

Adenosine acting through [A.sub.1] adenosine receptors (A1AR) has been shown previously to be required for the vasoconstriction elicited by high luminal NaCl concentrations at the macula densa (MD). The present experiments were performed to investigate a possible role of A1AR in MD control of renin secretion in conscious wild-type (WT) and A1AR-deficient mice. The intravenous injection of NaCl (5% body wt) reduced plasma renin concentration (PRC; ng ANG I*[ml.sup-1]*[h.sup.-1]) from 1,479 [+ or -] 129 to 711 [+ or -] 77 (P < 0.0001; n = 18) in WT mice but did not significantly change PRC in A1AR-/- mice (1,352 [+ or -] 168 during control vs. 1,744 [+ or -] 294 following NaCl; P = 0.19; n = 17). NaCl injections also caused a significant reduction in PRC in [[beta].sub.1]/[[beta].sub.2]-adrenergic receptor-/- mice (298 [+ or -] 47 vs. 183 [+ or -] 42; P = 0.03; n = 6). Injections of isotonic NaHC[O.sub.3] (5% body wt) elicited significant increases in PRC in both WT and A1AR-/- mice. NaCl as well as NaHC[O.sub.3] injections were accompanied by transient increases in blood pressure, heart rate, and activity that were similar in WT and A1AR-/- mice. The increase in PRC caused by an intraperitoneal injection of furosemide (40 mg/kg) was comparable in WT and A1AR-/- mice, and it was accompanied by similar transient increases in blood pressure, heart rate, and activity. Similarly, the stimulation of PRC caused by hydralazine was the same in WT and A1AR-/- mice. We conclude that the inhibition of renin secretion in response to an increase in NaCl at the MD requires A1AR and therefore appears to be adenosine dependent, whereas the stimulation of renin secretion during reductions in MD NaCl transport or arterial pressure does not require functional A1AR. At adenosine receptor; [[beta].sub.1]/[[beta].sub.2]-adrenergic receptor knockout; telemetry; furosemide; hydralazine; propranolol

Acute administration of loop diuretics like furosemide leads to a stimulation of renin secretion, an effect thought to result from inhibition of Na-K-2C1 cotransporter (NKCC2)-mediated salt transport at the luminal surface of the macula densa (MD). However, loop diuretics also inhibit NKCC1, the second isoform of the Na-K-2C1 cotransporter, with similar potency. In the present study, we examined the influence of furosemide on renin secretion in NKCC1-deficient mice to distinguish between effects of the loop diuretic involving NKCC2 and, by implication, the MD pathway, and effects that might occur via inhibition of NKCC 1. Baseline plasma renin concentration (PRC) was 1,212 [+ or -] 211 in NKCC1+/+ (n = 13) and 3,851 [+ or -] 579 ng ANG I*[ml.sup.-1]*[h.sup.-1] in NKCC1-/- mice (n = 14; P = 0.00024). Acute administration of furosemide (50 mg/kg i.p.) increased PRC significantly to 9,324 [+ or -] 1,018 ng ANG I*[m.sup.-1]*[h.sup.-1] in NKCCI+/+ (n = 13; P < 0.0001 compared with basal) and to 14,188 [+ or -] 2,274 ng ANG I*[ml.sup.-1]*[h.sup.-1] in NKCC1-/- mice [n = 14; P = 0.0002 compared with basal; P = 0.034 compared with wild-type (WT) plus furosemide]. Renin mRNA expression was about threefold higher in NKCCI-/- compared with WT mice. There was considerable recruitment of granular cells to upstream regions of afferent arterioles in NKCC1-/- mice. Patch-clamp studies in single juxtaglomerular granular (JG) cells from WT mice showed an ~10% increase in membrane capacitance during incubation with furosemide ([10.sup.-4] M), indicating a direct effect of the loop diuretic on renin secretion. No effect of furosemide on membrane capacitance was observed in JG cells from NKCC1-deficient mice. Furosemide ([10.sup.-3] M) significantly stimulated renin release from primary cultures of JG cells from WT mice, whereas no response was observed in NKCC1-/- mice. Our data suggest that a functional NKCC1 suppresses basal renin release, at least in part, through a direct effect on JG cells. NKCC1 knockout mice; plasma renin; renin mRNA; patch clamp; juxtaglomerular granular cells

The present study was undertaken to determine whether the severity of renal failure or hypertension in homozygous cyclooxygenase (COX)-2-deficient (COX-2-/-) mice affected by genetic background or gender. COX-2 deletion was introduced into three congenic genetic backgrounds, 129/Sv (129/COX-2-/-), C57/BL6 (C57/COX-2-/-), and BALB/c (BALB/COX-2-/-), by backcrossing the original mixed-background knockout mice with the respective inbred strains for 9 or 10 generations. Evaluation of the severity of hypertension and renal failure was performed in knockout and wild-type mice at the age of 2.5-3.5 mo. Blood pressure measured by tail-cuff plethysmography was significantly elevated in the male 129/COX-2-/-mice (165.8 [+ or -] 9.2 vs. 116 [+ or -] 5.1 mmHg, P < 0.05), and to a much lesser extent in the female 129/COX-2-/- mice (127.4 [+ or -] 3.3 vs. 102.4 [+ or -] 3.3), whereas it was unchanged in the C57- or BALB/ COX-2-/- mice regardless of gender. Urinary excretion of albumin, determined by EIA, was remarkably increased in the 129/COX-2-/(16.4 [+ or -] 4.1 vs. 0.16 [+ or -] 0.043 mg albumin/mg creatinine, P < 0.001), and to a lesser extent in the male C57/COX-2-/- mice (0.595 [+ or -] 0.416 vs. 0.068 [+ or -] 0.019). Albumin excretion was not elevated in the male BALB/COX-2-/- or in female COX-2-/- mice on any of the three genetic backgrounds. Histological analysis showed abundant protein casts, dilated tubules, and infiltration of inflammatory cells in the male 129/COX-2-/- mice, but not in COX-2-/- mice in other strains or gender. However, the presence of small glomeruli in the nephrogenic zone was observed in all strains of COX-2 knockout mice, regardless of genetic background and gender. Therefore, we conclude that the severity of hypertension and renal failure in COX-2-deficient mice is influenced by genetic background and gender, whereas the incomplete maturation of outer cortical nephrons appears to be independent of genetic background effects.

It is well established that cyclooxygenase-2 (COX-2) and the neuronal form of nitric oxide synthase (nNOS) are coexpressed in macula densa cells and that the expression of both enzymes is stimulated in a number of high-renin states. To further explore the role of nNOS and COX-2 in renin secretion, we. determined plasma renin activity in mice deficient in nNOS or COX-2. Plasma renin activity was significantly reduced in nNOS -/- mice on a mixed genetic background and in COX-2 -/- mice on either BALB/c or C57/BL6 congenic backgrounds. In additional studies, we accumulated evidence to show an inhibitory influence of [PGE.sub.2] on nNOS expression. In a cultured macula densa cell line, [PGE.sub.2] significantly reduced nNOS mRNA expression, as quantified by real-time RT-PCR. In COX-2 -/- mice, nNOS mRNA expression in the kidney, determined by real-time RT-PCR, was upregulated throughout the postnatal periods, ranging from postnatal day (PND) 3 to PND 60. The induction of nNOS protein expression and NOS activity in COX-2 -/- mice was localized to macula densa cells using immunohistochemistry and NADPH-diaphorase staining methods, respectively. Therefore, these findings reveal that the absence of either COX-2 or nNOS is associated with suppressed renin secretion. Furthermore, the inhibitory effect of PG[E.sub.2] on nNOS mRNA expression indicates a novel interaction between NO and prostaglandin-mediated pathways of renin regulation. juxtaglomerular apparatus; cyclooxygenase-2; neuronal nitric oxide synthase; real-time reverse transcriptase-polymerase chain reaction; plasma renin activity

Experiments were performed in neuronal (nNOS)--and endothelial nitric oxide synthase (eNOS)-deficient mice to study the role of nitric oxide (NO) in macula densa control of renin secretion in vivo and in the isolated, perfused mouse kidney. Acute and chronic administration of loop diuretics was used as a method to stimulate macula densa-mediated renin secretion. Increases in plasma renin concentration (PRC) in response to a 3-day infusion of bumetanide (50 mg*[kg.sup.-1]*[day.sup.-1]) or an acute injection of furosemide (50 mg/kg ip) were not markedly altered in nNOS-/-mice. Responses to furosemide were also maintained in eNOS-/-mice, but the administration of [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) markedly attenuated the PRC response to furosemide in these mice. In the isolated kidney preparation, bumetanide caused similar relative increases in renin secretion in kidneys of wild-type, nNOS-/-, and eNOS-/- mice. Bumetanide only marginally increased renin secretion in L-NAME-treated kidneys, hut the bumetanide effect was normalized by S-nitroso-N-acetyl-penicillamine. Basal PRC was significantly reduced in male nNOS-/- mice compared with nNOS+/+ (189 [+ or -] 28 vs. 355 [+ or -] 57 ng ANG I*[ml.sup.-1]*[h.sup.-1]; P = 0.017). There was no significant difference in PRC between eNOS+/+ and eNOS-/- mice. Basal renin secretion rates in perfused kidneys isolated from nNOS-/- or eNOS-/- mice were markedly reduced compared with wild-type controls. Our data suggest that NO generated by macula densa nNOS does not play a specific mediator role in macula densa-dependent renin secretion. However, NO independent of its exact source permits the macula densa pathway of renin secretion to function normally. plasma renin; isolated, perfused kidney; neuronal nitric oxide synthase knockout; endothelial nitric oxide synthase knockout; furosemide; bumetanide

Controversy exists regarding the effect of [A.sub.1] adenosine receptor (AR) activation in the kidney during ischemia and reperfusion (I/R) injury. We sought to further characterize the role of [A.sub.1] ARs in modulating renal function after UR renal injury using both pharmacological and gene deletion approaches in mice. [A.sub.1] AR knockout mice ([A.sub.1]KO) or their wild-type littermate controls ([A.sub.1]WT) were subjected to 30 min of renal ischemia. Some [A.sub.1]WT mice were subjected to 30 min of renal ischemia with or without pretreatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or 2-chrolo-cyclopentyladenosine (CCPA), selective [A.sub.1] AR antagonist and agonist, respectively. Plasma creatinine and renal histology were compared 24 h after renal injury. [A.sub.1]KO mice exhibited significantly higher creatinines and worsened renal histology compared with [A.sub.1]WT controls following renal I/R injury. [A.sub.1]WT mice pretreated with the [A.sub.1]AR antagonist or agonist demonstrated significantly worsened or improved renal function, respectively, after I/R injury. In addition, [A.sub.1]WT mice pretreated with DPCPX or CCPA showed significantly increased or reduced markers of renal inflammation, respectively (renal myeloperoxidase activity, renal tubular neutrophil infiltration, ICAM-I, TNF-[alpha], and IL-1[beta] mRNA expression), while demonstrating no differences in indicators of apoptosis. In conclusion, we demonstrate that endogenous or exogenous preischemic activation of [A.sub.1] ARs protects against renal I/R injury in vivo via mechanisms leading to decreased necrosis and inflammation. acute renal failure; inflammation; ischemic-repefusion injury

Adenosine is an ATP breakdown product that in most vessels causes vasodilatation and that contributes to the metabolic control of organ perfusion, i.e., to the match between oxygen demand and oxygen delivery. In the renal vasculature, in contrast, adenosine can produce vasoconstriction, a response that has been suggested to be an organ-specific version of metabolic control designed to restrict organ perfusion when transport work increases. However, the vasoconstriction elicited by an intravenous infusion of adenosine is only short lasting, being replaced within 1-2 min by vasodilatation. It appears that the steady-state response to the increase of plasma adenosine levels above normal resulting from the infusion is global renal vasorelaxation that is the result of A2AR activation in most parts of the renal vasculature, including larger renal arteries, juxtamedullary afferent arterioles, efferent arterioles, and medullary vessels. [A.sub.2]AR-mediated vasorelaxation is probably facilitated by endothelial receptors that cause the release of nitric oxide and other endothelial relaxing factors. In contrast, isolated perfused afferent arterioles of superficial and midcortical nephrons of rabbit and mouse, especially in their most distal segment at the entrance to the glomerulus, respond to adenosine with persistent vasoconstriction, indicating predominant or exclusive expression of [A.sub.1]AR. [A.sub.1]AR in afferent arterioles are selectively activated from the interstitial aspect of the vessel. This property can dissociate [A.sub.1]AR activation from changes in vascular adenosine concentration, a characteristic that is ideally suited for the role of renal adenosine as a paracrine factor in the control of glomerular function. adenosine receptors; vascular resistance; renal blood flow; endothelium

In the present experiments we examined the renovascular constrictor effects of ANG II in the chronic and complete absence of A1 adenosine receptors (A1AR) using mice with targeted deletion of the A1AR gene. Glomerular filtration rate (GFR) was not different between A1AR +/+ and A1AR -/- mice under control conditions (450.5 [+ or -] 60 vs. 475.2 [+ or -] 62.5 [micro]l/min) but fell significantly less in A1AR -/- mice during infusion of ANG II at 1.5 ng/min (A1AR +/+: 242 [+ or -] 32.5 [micro]l/min, A1AR -/-: 371 [+ or -] 42 [micro]l/min; P = 0.03). Bolus injection of 1, 10, and 100 ng of ANG II reduced renal blood flow and increased renal vascular resistance significantly more in A1AR +/+ than in A1AR -/- mice. Perfused afferent arterioles isolated from AIAR +/+ mice constricted in response to bath ANG II with an E[C.sub.50] of 1.5 [+ or -] 0.4 x [10.sup.-10] mol/l, whereas a right shift in the dose-response relationship with an E[C.sub.50] of 7.3 [+ or -] 1.2 x [10.sup.-10] mol/l (P < 0.05) was obtained in arterioles from A1AR -/- mice (P < 0.05). The expression of AT1A receptor mRNA was not different in kidney RNA from A1AR +/+ or A1AR -/- mice. We conclude that chronic A1AR deficiency diminishes the effectiveness of ANG II to constrict renal resistance vessels and to reduce GFR. renal blood flow; ultrasonic flowmeter; renal vascular resistance; glomerular filtration rate; perfused arterioles

Recent studies demonstrated that the influence of the macula densa on glomerular filtration is abolished in adenosine [A.sub.1] receptor ([A.sub.1]AR) knockout mice. Inasmuch as the macula densa not only regulates glomerular filtration but also controls the activity of the renin system, the present study aimed to determine the role of the [A.sub.1]AR in macula densa control of renin synthesis and secretion. Although a high-salt diet over 1 wk suppressed renin mRNA expression and renal renin content to similar degrees in [A.sub.1][AR.sup.+/+], [A.sub.1][AR.sup.+/-] , and [A.sub.1][AR.sup.-/-] mice, stimulation of Ren-1 mRNA expression and renal renin content by salt restriction was markedly enhanced in [A.sub.1][AR.sup.-/-] compared with wild-type mice. Pharmacological blockade of macula densa salt transport with loop diuretics stimulated renin expression in vivo (treatment with furosemide at 1.2 mg/day for 6 days) and renin secretion in isolated perfused mouse kidneys (treatment with 100 [mciro]M bumetanide) in all three genotypes to the same extent. Taken together, our data are consistent with the concept of a tonic inhibitory role of the [A.sub.1]AR in the renin system, whereas they indicate that the [A.sub.1]AR is not indispensable in macula densa control of the renin system. loop diuretics; low salt; high salt