Axial and cellular heterogeneity in electrolyte transport pathways along the thick ascending limb.

The thick ascending limb (TAL) extends from the border of the inner medulla to the renal cortex, thus ascending through regions with wide differences in tissue solute and electrolyte concentrations. Structural and functional differences between TAL cells in the medulla (mTAL) and the cortex (cTAL) would therefore be useful to adapt TAL transport function to a changing external fluid composition. While mechanisms common to all TAL cells play a central role in the reclamation of about 25% of the NaCl filtered by the kidney, morphological features, Na+ / K+ -ATPase activity, NKCC2 splicing and phosphorylation do vary between segments and cells. The TAL contributes to K+ homeostasis and TAL cells with high or low basolateral K+ conductances have been identified which may be involved in K+ reabsorption and secretion respectively. Although transport rates for HCO3- do not differ between mTAL and cTAL, divergent axial and cellular expression of H+ transport proteins in TAL have been documented. The reabsorption of the divalent cations Ca2+ and Mg2+ is highest in cTAL and paralleled by differences in divalent cation permeability and the expression of select claudins. Morphologically, two cell types with different cell surface phenotypes have been described that still need to be linked to specific functional characteristics. The unique external environment and its change along the longitudinal axis require an axial functional heterogeneity for the TAL to optimally participate in conserving electrolyte homeostasis. Despite substantial progress in understanding TAL function, there are still considerable knowledge gaps that are just beginning to become bridged.

Blood pressure, heart rate and tubuloglomerular feedback in A1AR-deficient mice with different genetic backgrounds.

AIM: Differences in genetic background between control mice and mice with targeted gene mutations have been recognized as a potential cause for phenotypic differences. In this study, we have used A1AR-deficient mice in a C57Bl/6 and SWR/J congenic background to assess the influence of background on the effect of A1AR-deficiency on cardiovascular and renal functional parameters. METHODS: In A1AR+/+ and A1AR-/- mice in C57Bl/6 and SWR/J congenic backgrounds, we assessed blood pressure and heart rate using radio-telemetry, plasma renin concentrations and tubuloglomerular feedback. RESULTS: We did not detect significant differences in arterial blood pressure (MAP) and heart rates (HR) between A1AR+/+ and A1AR-/- mice in either C57Bl/6, SWR/J or mixed backgrounds. MAP and HR were significantly higher in SWR/J than in C57Bl/6 mice. A high NaCl intake increased MAP in A1AR-/- mice on C57Bl/6 background while there was less or no salt sensitivity in the SWR/J background. No significant differences in plasma renin concentration were detected between A1AR-/- and A1AR+/+ mice in any of the strains. Tubuloglomerular feedback was found to be absent in A1AR-/- mice with SWR/J genetic background. CONCLUSIONS: While this study confirmed important differences between inbred mouse strains, we did not identify phenotypic modifications of A1AR-related effects on blood pressure, heart rate and plasma renin by differences in genetic background.

Annexin A1 modulates macula densa function by inhibiting cyclooxygenase 2.

Annexin A1 (ANXA1) exerts anti-inflammatory effects through multiple mechanisms including inhibition of prostaglandin synthesis. Once secreted, ANXA1 can bind to G protein-coupled formyl peptide receptors (Fpr) and activate diverse cellular signaling pathways. ANXA1 is known to be expressed in cells of the juxtaglomerular apparatus, but its relation to the expression of cyclooxygenase 2 (COX-2) in thick ascending limb and macula densa cells has not been elucidated. We hypothesized that ANXA1 regulates the biosynthesis of COX-2. ANXA1 abundance in rat kidney macula densa was extensively colocalized with COX-2 (95%). Furosemide, an established stimulus for COX-2 induction, caused enhanced expression of both ANXA1 and COX-2 with maintained colocalization (99%). In ANXA1-deficient mice, COX-2-positive cells were more numerous than in control mice (+107%; normalized to glomerular number; P < 0.05) and renin expression was increased (+566%; normalized to glomerular number; P < 0.05). Cultured macula densa cells transfected with full-length rat ANXA1 revealed downregulation of COX-2 mRNA (-59%; P < 0.05). Similarly, treatment with dexamethasone suppressed COX-2 mRNA in the cells (-49%; P < 0.05), while inducing ANXA1 mRNA (+56%; P < 0.05) and ANXA1 protein secretion. Inhibition of the ANXA-1 receptor Fpr1 with cyclosporin H blunted the effect of dexamethasone on COX-2 expression. These data show that ANXA1 exerts an inhibitory effect on COX-2 expression in the macula densa. ANXA1 may be a novel intrinsic modulator of renal juxtaglomerular regulation by inhibition of PGE(2) synthesis.

A(1) adenosine receptor-mediated PKC and p42/p44 MAPK signaling in mouse coronary artery smooth muscle cells.

The A(1) adenosine receptor (A(1)AR) is coupled to G(i)/G(o) proteins, but the downstream signaling pathways in smooth muscle cells are unclear. This study was performed in coronary artery smooth muscle cells (CASMCs) isolated from the mouse heart [A(1)AR wild type (A(1)WT) and A(1)AR knockout (A(1)KO)] to delineate A(1)AR signaling through the PKC pathway. In A(1)WT cells, treatment with (2S)-N(6)-(2-endo-norbornyl)adenosine (ENBA; 10(-5)M) increased A(1)AR expression by 150%, which was inhibited significantly by the A(1)AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (10(-6)M), but not in A(1)KO CASMCs. PKC isoforms were identified by Western blot analysis in the cytosolic and membrane fractions of cell homogenates of CASMCs. In A(1)WT and A(1)KO cells, significant levels of basal PKC-alpha were detected in the cytosolic fraction. Treatment with the A(1)AR agonist ENBA (10(-5)M) translocated PKC-alpha from the cytosolic to membrane fraction significantly in A(1)WT but not A(1)KO cells. Phospholipase C isoforms (betaI, betaIII, and gamma(1)) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-betaIII in A(1)WT CASMCs while having no effect in A(1)KO CASMCs. In A(1)WT cells, ENBA increased PKC-alpha expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-alpha inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A(1)AR stimulation by ENBA activates the PKC-alpha signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.

Distribution of cells expressing human renin-promoter activity in the brain of a transgenic mouse.

Renin plays a critical role in fluid and electrolyte homeostasis by cleaving angiotensinogen to produce Ang peptides. Whilst it has been demonstrated that renin mRNA is expressed in the brain, the distribution of cells responsible for this expression remains uncertain. We have used a transgenic mouse approach in an attempt to address this question. A transgenic mouse, in which a 12.2 kb fragment of the human renin promoter was used to drive expression of Cre-recombinase, was crossed with the ROSA26-lac Z reporter mouse strain. Cre-recombinase mediated excision of the floxed stop cassette resulted in expression of the reporter protein, beta-galactosidase. This study describes the distribution of beta-galactosidase in the brain of the crossed transgenic mouse. In all cases where it was examined the reporter protein was co-localized with the neuronal marker NeuN. An extensive distribution was observed with numerous cells labeled in the somatosensory, insular, piriform and retrosplenial cortices. The motor cortex was devoid of labeled cells. Several other regions were labeled including the parts of the amygdala, periaqueductal gray, lateral parabrachial nucleus and deep cerebellar nuclei. Overall the distribution shows little overlap with those regions that are known to express receptors for the renin-angiotensin system in the adult brain. This transgenic approach, which demonstrates the distribution of cells which have activated the human renin promoter at any time throughout development, yields a unique and extensive distribution of putative renin-expressing neurons. Our observations suggest that renin may have broader actions in the brain and may indicate a potential for interaction with the (pro)renin receptor or production of a ligand for non-AT(1)/AT(2) receptors.

AP214, an analogue of alpha-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality.

Sepsis remains a serious problem in critically ill patients with the mortality increasing to over half when there is attendant acute kidney injury. alpha-Melanocyte-stimulating hormone is a potent anti-inflammatory cytokine that inhibits many forms of inflammation including that with acute kidney injury. We tested whether a new alpha-melanocyte-stimulating hormone analogue (AP214), which has increased binding affinity to melanocortin receptors, improves sepsis-induced kidney injury and mortality using a cecal ligation and puncture mouse model. In the lethal cecal ligation-puncture model of sepsis, severe hypotension and bradycardia resulted and AP214 attenuated acute kidney injury of the lethal model with a bell-shaped dose-response curve. An optimum AP214 dose reduced acute kidney injury even when it was administered 6 h after surgery and it significantly improved blood pressure and heart rate. AP214 reduced serum TNF-alpha and IL-10 levels with a bell-shaped dose-response curve. Additionally; NF-kappaB activation in the kidney and spleen, and splenocyte apoptosis were decreased by the treatment. AP214 significantly improved survival in both lethal and sublethal models. We have shown that AP214 improves hemodynamic failure, acute kidney injury, mortality and splenocyte apoptosis attenuating pro- and anti-inflammatory actions due to sepsis.

Intracellular signalling pathways in the vasoconstrictor response of mouse afferent arterioles to adenosine.

AIMS: Adenosine causes vasoconstriction of afferent arterioles of the mouse kidney through activation of adenosine A(1) receptors and Gi-mediated stimulation of phospholipase C. In the present study, we further explored the signalling pathways by which adenosine causes arteriolar vasoconstriction. METHODS AND RESULTS: Adenosine (10(-7) M) significantly increased the intracellular calcium concentration in mouse isolated afferent arterioles measured by fura-2 fluorescence. Pre-treatment with thapsigargin (2 microM) blocked the vasoconstrictor action of adenosine (10(-7) M) indicating that release of calcium from the sarcoplasmic reticulum (SR), stimulated presumably by IP(3), is involved in the adenosine contraction mechanism of the afferent arteriole. In agreement with this notion is the observation that 2 aminoethoxydiphenyl borate (100 microM) blocked the adenosine-induced constriction whereas the protein kinase C inhibitor calphostin C had no effect. The calcium-activated chloride channel inhibitor IAA-94 (30 microM) inhibited the adenosine-mediated constriction. Patch clamp experiments showed that adenosine treatment induced a depolarizing current in preglomerular smooth muscle cells which was abolished by IAA-94. Furthermore, the vasoconstriction caused by adenosine was significantly inhibited by 5 microM nifedipine (control 8.3 +/- 0.2 microM, ado 3.6 +/- 0.6 microM, ado + nifedipine 6.8 +/- 0.2 microM) suggesting involvement of voltage-dependent calcium channels. CONCLUSION: We conclude that adenosine mediates vasoconstriction of afferent arterioles through an increase in intracellular calcium concentration resulting from release of calcium from the SR followed by activation of Ca(2+)-activated chloride channels leading to depolarization and influx of calcium through voltage-dependent calcium channels.

Role of A1 adenosine receptor in the regulation of coronary flow.

To determine whether A1 adenosine receptors (AR) participate in adenosine-induced changes of coronary flow, isolated hearts from A1AR(-/-) and A1AR(+/+) mice were perfused under constant pressure, and the effects of nonselective and selective agonists were examined. Adenosine, 5'-N-ethylcarboxamidoadenosine (NECA, nonselective), and the selective A2AAR agonist 2-2-carboxyethylphenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) augmented maximal coronary vasodilation in A1AR(-/-) hearts compared with A1AR(+/+) hearts. Basal coronary flow was increased (P < 0.05) in A1AR(-/-) hearts compared with A1AR(+/+) hearts: 2.548 +/- 0.1 vs. 2.059 +/- 0.17 ml/min. In addition, selective activation of A1AR with 2-chloro-N6-cyclopentyladenosine (CCPA) at nanomolar concentrations (1-100 nM) did not significantly change coronary flow; at higher concentrations, CCPA increased coronary flow in A1AR(-/-) and A1AR(+/+) hearts. Because deletion of A1AR increased basal coronary flow, it is speculated that this effect is due to removal of an inhibitory influence associated with A1AR. Adenosine and NECA at approximately EC50 (100 and 50 nM, respectively) increased coronary flow in A1AR(+/+) hearts to 177.86 +/- 8.75 and 172.72 +/- 17% of baseline, respectively. In the presence of the selective A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 50 nM), the adenosine- and NECA-induced increase in coronary flow in A1AR(+/+) hearts was significantly augmented to 216.106 +/- 8.35 and 201.61 +/- 21.89% of normalized baseline values, respectively. The adenosine- and NECA-induced increase in coronary flow in A1AR(-/-) hearts was not altered by DPCPX. These data indicate that A1AR may inhibit or negatively modulate coronary flow mediated by other AR subtypes (A2A and A2B).

Effect of apocynin treatment on renal expression of COX-2, NOS1, and renin in Wistar-Kyoto and spontaneously hypertensive rats.

Macula densa (MD) cells of the juxtaglomerular apparatus (JGA) synthesize type 1 nitric oxide synthase (NOS1) and type 2 cyclooxygenase (COX-2). Both nitric oxide (NO) and prostaglandins have been considered to mediate or modulate the control of renin secretion. Reactive oxygen species (ROS) produced locally by NADPH oxidase may influence NO bioavailability. We have tested the hypothesis that in hypertension elevated ROS levels may modify the expression of NOS1 and COX-2 in the JGA, thereby interacting with juxtaglomerular signaling. To this end, spontaneously hypertensive rats (SHR) and Wistar-Kyoto control rats (WKY) received the specific NADPH oxidase inhibitor, apocynin, during 3 wk. Renal functional and histochemical parameters, plasma renin activity (PRA), and as a measure of ROS activity, urinary isoprostane excretion (IP) were evaluated. Compared with WKY, IP levels in untreated SHR were 2.2-fold increased, and NOS1 immunoreactiviy (IR) of JGA 1.5-fold increased, whereas COX-2 IR was reduced to 35%, renin IR to 51%, and PRA to 7%. Apocynin treatment reduced IP levels in SHR to 52%, NOS1 IR to 69%, and renin IR to 62% of untreated SHR, whereas renin mRNA, COX-2 IR, glomerular filtration rate, PRA, and systolic blood pressure remained unchanged. WKY revealed no changes under apocynin treatment. These data show that NADPH oxidase is an important contributor to elevated levels of ROS in hypertension. Upregulation of MD NOS1 in SHR may have the potential of blunting the functional impact of ROS at the level of bioavailable NO. Downregulated COX-2 and renin levels in SHR are apparently unrelated to oxidative stress, since apocynin treatment had no effect on these parameters.

Reporter gene recombination in juxtaglomerular granular and collecting duct cells by human renin promoter-Cre recombinase transgene.

To assess the feasibility of using the renin promoter for expressing Cre recombinase in juxtaglomerular (JG) cells only, we generated five independent transgenic mouse lines (designated hRen-Cre) expressing Cre recombinase under control of a 12.2-kb human renin promoter. In the kidneys of adult mice Cre mRNA (RT-PCR) was found in the renal cortex, with Cre protein (immunohistochemistry) being localized in afferent arterioles and to a lower degree in interlobular arteries. Cre mRNA levels were regulated in a renin-typical fashion by changes in oral salt intake, water restriction, or isoproterenol infusion, indicating the presence of key regulatory elements within 12.2 kb of the 5'-flanking region of the human renin gene. hRen-Cre mice were interbred with both the ROSA26-EGFP and ROSA26-lacZ reporter strains to assess renin promoter activity from Cre-mediated excision of a floxed stop cassette and subsequent enhanced green fluorescent protein (EGFP) and beta-galactosidase (beta-gal) detection. In adult mice, beta-gal staining and EGFP were observed in afferent arterioles and interlobular arteries, overlapping with Cre protein expression. In addition, intense beta-gal staining was found in cortical and medullary collecting ducts where Cre expression was minimal. In embryonic kidneys, beta-gal staining was detected in the developing collecting duct system beginning at embryonic day 12, showing substantial activity of the human renin promoter in the branching ureteric bud. Our data indicate that besides its well-known activity in JG cells and renal vessels the human renin promoter is transiently active in the collecting duct system during kidney development, complicating the use of this approach for JG cell-specific excision of floxed targets.

Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice.

Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A(1) receptor (A(1)AR)-knockout (A(1)KO) mice and their wild-type (A(1)WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle alpha-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was approximately 91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A(1)WT and A(1)KO mice. Furthermore, the A(1)AR was characterized by Western blot analysis using an A(1)AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.

Reduced autoregulatory effectiveness in adenosine 1 receptor-deficient mice.

Adjustments of renal vascular resistance in response to changes in blood pressure are mediated by an interplay between the myocyte-inherent myogenic and the kidney-specific tubuloglomerular feedback (TGF) mechanisms. Using mice with deletion of the A(1) adenosine receptor (A1AR) gene, we tested the prediction that the absence of TGF, previously established to result from A1AR deficiency, is associated with a reduction in the efficiency of autoregulation. In anesthetized wild-type (A1AR+/+) and A1AR-deficient mice (A1AR-/-), glomerular filtration rate (GFR) and renal blood flow (RBF) were determined before and after reducing renal perfusion pressure through a suprarenal aortic clamp. In response to a blood pressure reduction by 15.9 +/- 1.34 mmHg in A1AR-/- (n = 9) and by 14.2 +/- 0.9 mmHg in A1AR+/+ mice (n = 8; P = 0.31), GFR fell by 187.9 +/- 37 mul/min and by 72.3 +/- 10 mul/min in A1AR-/- and A1AR+/+ mice, respectively (P = 0.013). Similarly, with pressure reductions of 14.8 +/- 1.1 and 13.3 +/- 1.5 mmHg in A1AR-/- (n = 9) and wild-type mice (n = 8), respectively (P = 0.43), RBF fell by 0.17 +/- 0.02 ml/min in A1AR-/- mice and by only 0.08 +/- 0.02 ml/min in wild-type animals (P = 0.0039). Autoregulatory indexes for both GFR and RBF were significantly higher in A1AR-/- compared with A1AR+/+ mice, indicating reduced regulatory responsiveness in the knockout animals. We conclude that autoregulation of renal vascular resistance is less complete in A1AR-deficient mice, an effect that is presumably related to absence of TGF regulation in these animals.

Vasoconstrictor and vasodilator effects of adenosine in the mouse kidney due to preferential activation of A1 or A2 adenosine receptors.

The present experiments in mice were performed to determine the steady-state effects of exogenous adenosine on the vascular resistance of the whole kidney, of superficial blood vessels, and of afferent arterioles. The steady-state effect of an intravenous infusion of adenosine (5, 10, and 20 microg/min) in wild-type mice was vasodilatation as evidenced by significant reductions of renal and superficial vascular resistance. Resistance decreases were augmented in adenosine 1 receptor (A1AR) -/- mice. Renal vasodilatation by the A2aAR agonist CGS 21680A [2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamido-adenosine hydrochloride] (0.25, 0.5, and 1 microg/kg/min) and inhibition of adenosine-induced relaxation by the A2aAR antagonist ZM-241385 [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol] (20 mg/kg) suggests that the reduction of renovascular resistance was largely mediated by A2aAR. After treatment with Nomega-nitro-L-arginine methyl ester (L-NAME) adenosine was unable to alter superficial blood flow and resistance significantly indicating that adenosine-induced dilatation is NO-dependent. Absence of a dilatory effect in endothelial nitric-oxide synthase (NOS) -/- mice suggests endothelial NOS as the source of NO. When infused into the subcapsular interstitium, adenosine reduced superficial blood flow through A1AR activation. Adenosine (10(-7) M) constricted isolated perfused afferent arterioles when added to the bath but not when added to the luminal perfusate. Luminal adenosine caused vasoconstriction in the presence of L-NAME or the A2AR antagonist 3,7-dimethyl-1-(2-propynyl)xanthine. Our data show that global elevation of renal adenosine causes steady-state vasorelaxation resulting from adenosine 2 receptor (A2AR)-mediated generation of NO. In contrast, selective augmentation of adenosine around afferent arterioles causes persistent vasoconstriction, indicating A1AR dominance. Thus, adenosine is a renal constrictor only when it can interact with afferent arteriolar A1AR without affecting the bulk of renal A2AR at the same time.

Role of A1 adenosine receptors in regulation of vascular tone.

The vascular response to adenosine and its analogs is mediated by four adenosine receptors (ARs), namely, A(1), A(2A), A(2B), and A(3). A(2A)ARs and/or A(2B)ARs are involved in adenosine-mediated vascular relaxation of coronary and aortic beds. However, the role of A(1)ARs in the regulation of vascular tone is less well substantiated. The aim of this study was to determine the role of A(1)ARs in adenosine-mediated regulation of vascular tone. A(1)AR-knockout [A(1)AR((-/-))] mice and available pharmacological tools were used to elucidate the function of A(1)ARs and the impact of these receptors on the regulation of vascular tone. Isolated aortic rings from A(1)AR((-/-)) and wild-type [A(1)AR((+/+))] mice were precontracted with phenylephrine, and concentration-response curves for adenosine and its analogs, 5'-N-ethyl-carboxamidoadenosine (NECA, nonselective), 2-chloro-N(6)-cyclopentyladenosine (CCPA, A(1)AR selective), 2-(2-carboxyethyl)phenethyl amino-5'-N-ethylcarboxamido-adenosine (CGS-21680, A(2A) selective), and 2-chloro-N(6)-3-iodobenzyladenosine-5'-N-methyluronamide (Cl-IBMECA, A(3) selective) were obtained to determine relaxation. Adenosine and NECA (0.1 microM) caused small contractions of 13.9 +/- 3.0 and 16.4 +/- 6.4%, respectively, and CCPA at 0.1 and 1.0 microM caused contractions of 30.8 +/- 4.3 and 28.1 +/- 3.9%, respectively, in A(1)AR((+/+)) rings. NECA- and CCPA-induced contractions were eliminated by 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, selective A(1)AR antagonist). Adenosine, NECA, and CGS-21680 produced an increase in maximal relaxation in A(1)AR((-/-)) compared with A(1)AR((+/+)) rings, whereas Cl-IBMECA did not produce contraction in either A(1)AR((+/+)) or A(1)AR((-/-)) rings. CCPA-induced contraction at 1.0 microM was eliminated by the PLC inhibitor U-73122. These data suggest that activation of A(1)ARs causes contraction of vascular smooth muscle through PLC pathways and negatively modulates the vascular relaxation mediated by other adenosine receptor subtypes.

Plasma renin in mice with one or two renin genes.

AIM: In the present study we have investigated whether the presence of a second renin gene exerts an overriding influence on plasma renin such that mice with two renin genes have consistently higher renin levels than mice with only one renin gene. METHODS: Plasma renin was determined as the rate of angiotensin I generation using a radioimmunoassay (RIA) kit with (plasma renin concentration, PRC) or without (plasma renin activity, PRA) the addition of purified rat angiotensinogen as substrate. RESULTS: In male 129SvJ, DBA/2 and Swiss Webster mice, strains possessing both Ren-1 and Ren-2, PRC (ng Ang I mL(-1) h(-1)) averaged 178 +/- 36, 563 +/- 57 and 550 +/- 43 while PRA was 2.9 +/- 0.5, 3.6 +/- 0.8 and 7.8 +/- 1.2. In male C57BL/6, C3H and BALB/c mice that express only Ren-1, PRC averaged 426 +/- 133, 917 +/- 105 and 315 +/- 72, and PRA was 3.4 +/- 1.0, 6.9 +/- 1.7 and 4.5 +/- 1.2. In the two renin gene A1AR-/- mice compared with the one renin gene A1AR+/+, PRC averaged 538 +/- 321 and 415 +/- 159 while PRA averaged 3.2 +/- 1.1 and 4.4 +/- 1.4 ng Ang I mL(-1) h(-1). Aldosterone levels showed no significant differences between one renin (C57BL/6, C3H and BALB/c) and two renin (129SvJ, DBA/2 and Swiss Webster) gene mice. Furthermore, by quantitative real-time polymerase chain reaction (RT-PCR) we found no correlation between the number of renin genes and whole kidney renin mRNA levels from one and two renin gene mice. CONCLUSION: Our data show that baseline plasma renin is not systematically higher in mice with two renin genes than in one renin gene mice. Thus, the presence of a second renin gene does not seem to be a major determinant of differences in PRC between different mouse strains.

Compensation of proximal tubule malabsorption in AQP1-deficient mice without TGF-mediated reduction of GFR.

AIM: By crossing aquaporin 1 (AQP1)-/- and adenosine 1 receptor (A1AR)-/- mice, we generated an animal model that combines a proximal tubular absorption defect with absence of tubuloglomerular feedback (TGF) regulation of glomerular filtration rate (GFR). The aim of studies in these animals was to determine whether a TGF-induced reduction of GFR is a prerequisite for preventing potentially fatal fluid losses. METHODS AND RESULTS: In contrast to AQP1 deficient mice, AQP1/A1AR-/- mice were found to have a normal GFR. TGF responses were abolished in these animals, in contrast to AQP1-/- mice in which TGF responses of single nephron glomerular filtration rate (SNGFR) were left-shifted. Proximal tubule fluid absorption in AQP1/A1AR-/- mice was reduced to levels previously reported for AQP1-/- mice. However, SNGFR was significantly higher in AQP1/A1AR-/- than AQP1-/- mice (10.6 +/- 0.8 nL min(-1) vs. 5.9 +/- 0.7 nL min(-1)). As a consequence of the normal GFR and the reduced proximal reabsorption distal fluid delivery was markedly higher in the double knockout compared with normal or AQP1-/- mice (5.5 +/- 0.5 nL min(-1) vs. 2.35 +/- 0.3 nL min(-1) in AQP1-/-). Despite the approximate doubling of distal fluid and Cl delivery, AQP1/A1AR-/- mice have a normal salt excretion, normal arterial blood pressure, and only a small increase in plasma renin concentration. CONCLUSION: The ability to compensate for proximal tubule malabsorption without a TGF-induced reduction of GFR attests to a remarkable adaptability of distal tubule transport mechanisms.

Functional consequences at the single-nephron level of the lack of adenosine A1 receptors and tubuloglomerular feedback in mice.

Mice deficient for adenosine A1 receptors (A1AR) lack tubuloglomerular feedback (TGF). In vivo micropuncture experiments were performed under anesthesia in A1AR-deficient and wild-type littermate mice to study the effects of chronic absence of A1AR on fluid and Na(+) reabsorption along the nephron, as well as the functional consequences at the single-nephron level of the lack TGF. Evidence is provided for an A1AR-mediated tonic inhibition of Na(+) reabsorption in a water-impermeable segment of the loop of Henle, possibly the thick ascending limb. In contrast, proximal tubular reabsorption of fluid, Na(+) and K(+) was unaffected by the chronic absence of A1AR. Experiments in which artificial tubular fluid was added to free-flowing late-proximal tubules demonstrated an essential role of A1AR/TGF in the stabilization of fluid and Na(+) delivery to the distal nephron. Further, the occurrence of spontaneous oscillations of hydrostatic pressure in proximal tubule ( P(PT)) at a frequency of about 32 mHz depended on intact A1AR/TGF. In comparison, the normal, stabilizing reduction in P(PT) following the initial rise in P(PT) during sustained small increases in proximal tubular flow rate does not require A1AR/TGF; TGF-independent mechanisms appear to compensate in this regard for a lack of TGF under physiological conditions and the lack of TGF is unmasked only when supraphysiological flow rates overwhelm TGF-independent compensation.

Cyclooxygenase cloning in dogfish shark, Squalus acanthias, and its role in rectal gland Cl secretion.

The present studies were carried out with the aims to determine the cDNA sequence for cyclooxygenase (COX) in an elasmobranch species and to study its role in regulation of chloride secretion in the perfused shark rectal gland (SRG). With the use of long primers (43 bp) derived from regions of homology between zebrafish and rainbow trout COX-2 genes, a 600-bp product was amplified from SRG and was found to be almost equally homologous to mammalian COX-1 and COX-2 (65%). The full-length cDNA sequence was obtained by 5'-RACE and by analyzing an EST clone generated by the EST Project of the Mt. Desert Island Biological Laboratory Marine DNA Sequencing Center. The longest open reading frame encodes a 593-amino acid protein that has 68 and 64% homology to mammalian COX-1 and COX-2, respectively. The gene and its protein product is designated as shark COX (sCOX). The key residues in the active site (Try(385), His(388), and Ser(530)) are conserved between the shark and mammalian COX. sCOX contains Val(523) that has been shown to be a key residue determining the sensitivity to COX-2-specific inhibitors including NS-398. The mRNA of sCOX, detected by RT-PCR, was found in all tissues tested, including rectal gland, kidney, spleen, gill, liver, brain, and heart, but not in fin. In the perfused SRG, vasoactive intestinal peptide (VIP) at 5 nM induced rapid and marked Cl(-) secretion (basal: <250 microeq x h(-1) x g(-1); peak response: 3,108 +/- 479 microeq x h(-1) x g(-1)). In the presence of 50 microM NS-398, both the peak response (2,131 +/- 307 microeq x h(-1) x g(-1)) and the sustained response to VIP were significantly reduced. When NS-398 was removed, there was a prompt recovery of chloride secretion to control values. In conclusion, we have cloned the first COX in an elasmobranch species (sCOX) and shown that sCOX inhibition suppresses VIP-stimulated chloride secretion in the perfused SRG.

Sodium transport deficiency and sodium balance in gene-targeted mice.

Animals with induced or natural null mutations in renal NaCl and water transporter genes provide a powerful tool to study the physiological mechanisms that enable the kidney to optimize the match between glomerular filtration rate and tubular reabsorption. Deficiencies in the Na/H exchanger NHE3 and in the water channel aquaporin 1 (AQP1) cause reductions in proximal fluid absorption which are accompanied by proportionate decrements in glomerular filtration rate (GFR). Compensation of the transport defect by a reduction in filtered load is so efficient that clinically symptomatic Na losses are not observed in either NHE3 or AQP1 deficient animals. On the other hand, severe syndromes of salt wasting are caused by loss of function of the Na,K,2Cl-cotransporter (NKCC2) in the thick ascending limb, or of the epithelial Na channel (ENaC) the collecting duct indicating that the severity of Na dysregulation is unrelated to the basal absorption of NaCl in a given nephron segment. In these states, the increased delivery of Na to downstream segments is not monitored by a sensor linked to the site of filtrate formation. In the absence of adaptations in the filtered load intrarenal compensation of a circumscribed NaCl malabsorption by adjustment of NaCl transport in other nephron segments is sometimes insufficient, particularly in the immature kidney of the newborn.