Low plasma renin and reduced renin secretory responses to acute stimuli in conscious COX-2-deficient mice.

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 microg), or quinaprilate (50 microg) 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., DeltaPRC in ng ANG I x ml(-1) x h(-1) caused by furosemide, isoproterenol, hydralazine, quinaprilate, or candesartan 4,699 +/- 544, 3,534 +/- 957, 2,522 +/- 369, 9,453 +/- 1,705, 66,455 +/- 21,938 in 129J WT, and 201 +/- 78, 869 +/- 275, 140 +/- 71, 902 +/- 304, 2,660 +/- 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 (DeltaPRC 201 +/- 78 before and 15,984 +/- 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.

Regulation of renin in mice with Cre recombinase-mediated deletion of G protein Gsalpha in juxtaglomerular cells.

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 Gsalpha in juxtaglomerular granular (JG) cells. Compared with wild-type animals, mice with conditional Gsalpha 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, Gsalpha-deficient mice had reduced arterial blood pressure, reduced levels of aldosterone, and a low glomerular 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(2) or isoproterenol was eliminated. Unexpectedly, Gsalpha 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 Gsalpha-mediated signal transduction is essential and nonredundant in the control of renin synthesis and release.

Adenosine as a mediator of macula densa-dependent inhibition of renin secretion.

Adenosine acting through A(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 x ml(-1) x h(-1)) from 1,479 +/- 129 to 711 +/- 77 (P < 0.0001; n = 18) in WT mice but did not significantly change PRC in A1AR-/- mice (1,352 +/- 168 during control vs. 1,744 +/- 294 following NaCl; P = 0.19; n = 17). NaCl injections also caused a significant reduction in PRC in beta(1)/beta(2)-adrenergic receptor-/- mice (298 +/- 47 vs. 183 +/- 42; P = 0.03; n = 6). Injections of isotonic NaHCO(3) (5% body wt) elicited significant increases in PRC in both WT and A1AR-/- mice. NaCl as well as NaHCO(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.

Epithelial COX-2 expression is not regulated by nitric oxide in rodent renal cortex.

In the adult rodent kidney cortex, cyclooxygenase-2 (COX-2), NO synthase (NOS1), and renin synthesis change in parallel on alterations in distal tubular NaCl concentration, and their products in part may mutually determine synthesis and activity of these enzymes. Epithelial NO synthesis has been postulated to exert a stimulatory role on COX-2 expression. Changes in COX-2 and NOS1 may be assessed histochemically by determining changes in the number of positive cells. In rat, macula densa and adjacent cells may co-express COX-2 and NOS1, whereas cell groups of the upstream thick ascending limb (cTAL) express COX-2 alone. We have tested whether the stimulation of COX-2 expression by short- and long-term unilateral renal artery stenosis, low salt, and furosemide treatment depends on co-expression of NOS1. These conditions produced significant respective increases (40% to 351%, P<0.05) in the number of COX-2 immunoreactive cells, regardless of whether NOS1 was present or not, suggesting that co-expression of NOS1 is not necessary to produce these changes. Under high-salt conditions, analogous though inverse changes were recorded (-62% to -73%, P<0.05). In mice with genetic deletion of NOS1, low- and high-salt diets caused similar changes of COX-2 immunoreactivity (106% and -52%, P<0.05) than those seen in wild-type mice (43% and -78%, P<0.05). We conclude that alterations of distal tubular NaCl concentration and presumably NaCl transport induce changes in epithelial COX-2 expression that does not depend on presence of co-expressed NOS1. It therefore seems unlikely that NO is part of a signal transduction chain between tubular chloride sensing and the modulating effects of prostaglandins in tubulo-vascular information transfer.

Feedback control of glomerular vascular tone in neuronal nitric oxide synthase knockout mice.

For further elucidation of the role of neuronal nitric oxide synthase (nNOS) in macula densa (MD) cells, experiments were performed in anesthetized nNOS knockout mice (nNOS -/-). At comparable levels of arterial BP, renal blood flow was not significantly different between nNOS +/+ and nNOS -/- (1.7 +/- 0.2 versus 1.4 +/- 0.1 ml/min), and autoregulation of renal blood flow was maintained to a pressure level of approximately 85 mmHg in both groups of mice (n = 6 in each group). The fall in proximal tubular stop-flow pressure in response to an increase in loop of Henle perfusion rate from 0 to 30 nl/min was comparable in nNOS +/+ and -/- mice (40.7 +/- 1.6 to 32 +/- 2 mmHg versus 40.6 +/- 1.6 to 31.6 +/- 2 mmHg; not significant; n = 13 versus 18 nephrons). Luminal application of the nonselective NOS inhibitor nitro-L-arginine (10(-3) and 10(-2) M) enhanced the perfusion-dependent fall in stop-flow pressure in nNOS +/+ (7 +/- 1 to 13 +/- 2 mmHg; P < 0.05) but not in nNOS -/- (7 +/- 1 to 8 +/- 1 mmHg; not significant) mice. nNOS -/- mice exhibited a lower nephron filtration rate, compared with nNOS +/+, during free-flow collections from early distal tubules (influence of MD intact, 7 +/- 0.7 versus 10.9 +/- 1 nl/min; P = 0.002) but not from late proximal tubule (influence of MD minimized, 10.1 +/- 1 versus 11.7 +/- 1 nl/min; not significant; n = 16 nephrons). Distal Cl concentration and fractional absorption of fluid or chloride up to the early distal tubule was not different between nNOS -/- and +/+ mice. The data indicate that nNOS in MD tonically attenuates the GFR-lowering influence of ambient luminal NaCl, which may serve to increase the fluid and electrolyte load to the distal tubule, consistent with a role of MD nNOS in tubuloglomerular feedback resetting.

Angiotensin II blockade causes acute renal failure in eNOS-deficient mice.

Compared with wild-type mice, adult endothelial nitric oxide synthase (eNOS) knockout mice (eight months of age) have increased blood pressure (BP) (126±9 mmHg vs. 100±4 mmHg), and an increased renal vascular resistance (155±16 vs. 65±4 mmHg.min/ml). Renal vascular resistance responses to i.v. administration of noradrenaline were markedly enhanced in eNOS knockout mice. Glomerular filtration rate (GFR) of anaesthetised eNOS -/- mice was 324±57 µl/min gKW, significantly lower than the GFR of 761±126 µl/min.gKW in wild-type mice. AT1-receptor blockade with i.v. candesartan (1-1.5 mg/kg) reduced arterial blood pressure and renal vascular resistance, and increased renal blood flow (RBF) to about the same extent in wild-type and eNOS -/- mice. Candesartan did not alter GFR in wild-type mice (761±126 vs. 720±95 µl/min.gKW), but caused a marked decrease in GFR in eNOS -/- mice (324.5±75.2 vs. 77±18 µl/min.gKW). A similar reduction in GFR of eNOS deficient mice was also caused by angiotensin-converting enzyme (ACE) inhibition. Afferent arteriolar granularity, a measure of renal renin expression, was found to be reduced in eNOS -/- compared with wild-type mice. In chronically eNOS-deficient mice, angiotensin II (Ang II) is critical for maintaining glomerular filtration pressure and GFR, presumably through its effect on efferent arteriolar tone.

Mouse beta(6) integrin sequence, pattern of expression, and role in kidney development.

Integrins mediate cell-cell and cell-extracellular matrix interactions and play key roles in development. beta(6) integrin expression has been demonstrated in human fetal kidney at a higher level than in the adult, making beta(6) integrin a marker of interest for the study of development of the nephron. The aims of this study were to determine the cDNA sequence for the mouse beta(6) integrin and to characterize beta(6) integrin expression in the developing mouse kidney. Two embryonic mouse kidney cDNA libraries were screened, and the coding region was sequenced. The mouse beta(6) nucleotide coding region sequence shows 82% nucleotide identity to the human sequence. The putative amino acid sequence has 89.5% identity to human beta(6) integrin and contains many conserved domains. By reverse transcription-PCR, beta(6) integrin mRNA expression is very low at 11 d of gestation in the mouse, increases dramatically by E14 and E17 (20-fold, normalized for increases in ss actin), and plateaus by 2 wk of age. beta(6) integrin expression is induced 15- to 20-fold after 5 d in metanephric explant culture. Reverse transcription-PCR of adult rat microdissected nephron segments demonstrates ss(6) integrin mRNA expression in proximal tubule, cortical thick ascending limb, distal nephron segments (inner and outer medullary collecting ducts), and macula densa-containing segments. Lectin-peroxidase and in situ colocalization studies demonstrated expression of ss(6) integrin mRNA in developing proximal tubules and thick ascending limb. Culture of mouse metanephric kidneys with antisense oligonucleotides to beta(6) integrin resulted in inhibition of ureteric bud branching and complete lack of mesenchyme condensation. These studies demonstrate a high homology between the human and mouse beta(6) integrin sequence, a different pattern of expression in the developing mouse kidney compared with the primate kidney, and abnormal metanephric development in culture in the absence of beta(6) integrin. These findings suggest an important role for beta(6) integrin in normal development of the mouse kidney.