The overall goal of our research is to contribute to understanding the role of the kidneys in the maintenance of body fluid volume and arterial blood pressure. Specifically, we study the renal mechanisms responsible for the match between glomerular filtration of plasma and tubular salt and fluid reabsorption that is ultimately responsible for the precise balance between salt intake and excretion. One of the key mechanisms that mediates between renal absorption and filtration function is the so-called tubuloglomerular feedback, a regulatory pathway in which a measure of tubular reabsorptive capacity serves as the signal to alter glomerular filtration. 1. JGA-mediated effects are sensitive to loop diuretics suggesting that NaCl transport through the NKCC2 cotransporter in the apical membrane of macula densa (MD) cells may be critical for eliciting JGA dependent responses. However, loop diuretics exert a host of secondary effects so that the specificity of this action and its mechanism have remained unclear. NKCC2 is expressed along the thick ascending limb (TAL) of the loop of Henle in three isoforms representing splice variants of a single gene. As previously shown by RT-PCR and in situ hybridization, the B isoform of NKCC2 is expressed in MD cells, the A isoform in both cortical and medullary TAL, and the F isoform in medullary TAL exclusively. To produce isoform-specific NKCC2 knockout mice we have inserted a stop codon into either the B, A, or F region of exon 4 of the NKCC2 gene by homologous recombination. After obtaining chimeric mice carrying either the B, A, or F isoform mutation we have bred the B and A isoform mutation to homozygosity while we are encountering unexpected difficulties to obtain homozygous F mutants. Both NKCC2A-/- and NKCC2B-/- mice were viable and showed no morphological abnormalities. Ambient urine osmolarity (mosm/l) was reduced in both NKCC2A-/- (1236 vs. 1673, p=.02) and NKCC2B-/- mice (1401 vs. 1890, p=.0004). Basal plasma renin concentration (PRC; ng Ang I/ml/hr) tended to be lower in NKCC2A-/- compared to WT (621 vs. 1211, p=.061), and was reduced in NKCC2B-/- mice (1040 vs. 1425, p=.0058). A low Na diet (0.003% NaCl) increased PRC in both NKCC2B+/+ and NKCC2B-/- mice. Maximum tubuloglomerular feedback (TGF) responses were not different between NKCC2B-/- and +/+ mice, but TGF curves were right-shifted. Cl absorption along loops of Henle of NKCC2B-/- mice significantly reduced compared to wild type. It appears that the loss of either NKCC2B or NKCC2A is partially compensated for by the remaining isoform and therefore does not dramatically alter overall loop of Henle or MD sensing functions. Mice lacking both NKCC2A and NKCC2B are needed to clarify the role of NKCC2 in MD control of preglomerular resistance and renin secretion, and we have begun to make a construct that would achieve this. 2. Renin formation in granular cells of the juxtaglomerular apparatus plays a critical role in the maintenance of body fluid volume and arterial blood pressure. There is evidence that agonists signaling through Gsalpha-coupled receptors are important mediators of some the pathways mediating renin secretion. To study the consequences of eliminating JGA regulation of renin secretion, we have used Cre-Lox based strategies to delete the Gsalpha-cAMP signaling pathway in renin-producing JG cells. In one of these approaches, we have crossed mice in which cre-recombinase under control of the Ren1d renin promoter has been introduced into JG cells by recombination in stem cells with mice in which Gsalpha is flanked by LoxP sites (generated by Drs. Weinstein and Chen at NIDDK). Ren1d-Cre/GsF.F mice were viable and showed no obvious anatomical abnormalities. Cre-mediated DNA recombination and inactivation of Gsalpha was found in the kidney cortex and in isolated JG cells where Gsalpha protein was reduced to about 25% of appropriate controls. Basal plasma renin concentration (PRC; ng AngI/ml/hr) was extremely low, and it did not significantly change with the administration of furosemide, isoproterenol, or hydralazine, interventions that cause marked elevations of PRC in wild type mice. In addition to JG cells, Gs???ninactivation was observed in the renal medulla as well as in other tissues, most notably the adrenal gland. In agreement with medullary Gsalpha deficiency, Ren1d-cre/GsF.F mice had a marked and AVP-resistant concentrating defect. We conclude that ambient as well as macula densa- and baroreceptor-dependent renin release is dominated by Gs-coupled receptor signaling. Thus, cyclic AMP appears to be the major intracellular messenger regulating renin synthesis and release. However, use of the renin promoter to express Cre-recombinase does not seem to achieve JG cell-specific elimination of floxed targets. 3. In a continued pursuit to clarifying the role of adenosine in juxtaglomerular signaling, we have performed studies to examine the role of A1AR in MD control of renin secretion in conscious mice. Furosemide (40 mg/kg i.p.) was used to reduce MD NaCl transport thereby stimulating renin secretion, and i.v. injections of isotonic NaCl (5% BW) were used to increase MD [NaCl] thereby inhibiting renin secretion. Furosemide caused plasma renin concentration (PRC, ng angiotensin I/ml/hr) to increase to the same extent in WT (5.4fold) and in A1AR-/- mice (4.8fold). The response to furosemide was not the result of sympathetic stimulation since furosemide caused a comparable increase of PRC in beta1/beta2-adrenergic receptor double knockout mice. Administration of NaCl, by providing an inhibitory signal to the macula densa, reduced PRC in male and female wild type mice, but it had no inhibitory effect in A1AR-/- mice. NaCl injection also reduced PRC in beta-adrenergic receptor-deficient mice. In contrast to NaCl, isotonic NaHCO3 (5% BW) increased PRC in both WT and A1AR-/- mice. These data indicate that the inhibition of renin secretion in response to an increase in [NaCl] at the MD requires A1AR and is therefore adenosine-dependent whereas the stimulation of renin secretion during reductions in [NaCl] is A1AR-independent suggesting a role for a mediator other than adenosine. Since substantial experimental evidence supports the concept that prostaglandins (PGs) generated by COX-2 in the juxtaglomerular apparatus (JGA) play an important role as a stimulatory mediator in macula densa (MD) control of renin secretion we determined the response of plasma renin concentration (PRC) to furosemide (40 mg/kg i.p.) and to hydralazine (2 mg/kg i.p.) in conscious wild type (WT) and COX-2-deficient (COX-/-) mice. Basal PRC was significantly higher in WT than COX-/- mice. Furosemide caused a marked increase of PRC in wild type mice, but its effect was greatly attenuated in COX2-/- mice. Similarly, while hydralazine increased PRC in wild type animals, its effect was reduced in COX-/- mice. As measured by radiotelemetry, furosemide did not significantly alter blood pressure in the period of observation whereas hydralazine reduced blood pressure by about 25 mm Hg. Our data show that the chronic absence of COX-2 causes a severe insufficiency to synthetize renin and therefore generates a chronic low renin state. This is associated with a reduced responsiveness of renin release to both MD and baroreceptor stimulation. A COX-2 product, presumably PGE2, appears to be critical for basal renin synthesis and release as well as for MD- and baroreceptor-dependent stimulation of renin secretion.
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