1. To address the question whether selective deletion of A1AR from smooth muscle cells is sufficient to abolish TGF responsiveness we have determined TGF response magnitude in mice in which vascular A1AR deletion was achieved using the loxP recombination approach with cre recombinase being controlled by a smooth muscle actin promoter (SmCre/A1ARff). Effective vascular deletion of A1AR was affirmed by absence of vasoconstrictor responses to adenosine or the A1AR agonist cyclohexyl adenosine (CHA) in microperfused afferent arterioles. TGF responses in SmCre/A1ARff mice were found to be significantly reduced, but not abolished. Maintenance of residual TGF activity despite absence of A1AR-mediated responses in AA suggests participation of extravascular A1AR in TGF. Support for this notion comes from the observation that deletion of A1ARff by nestin-driven cre recombinase caused an identical TGF response reduction while arteriolar responsiveness to A1AR activation was reduced, but not abolished. A1AR on arteriolar smooth muscle cells are primarily responsible for TGF activation, but A1AR on extravascular cells, perhaps mesangial cells, appear to contribute to the TGF response. 2. While previous evidence has indicated that the A1AR ligand adenosine is at least in part derived from the extracellular metabolism of nucleotides transmembrane movement of adenosine mediated by the widely distributed equilibrative nucleoside transporter 1 (ENT1) may also play a modifying role. To study this possibility we have assessed JGA function in ENT1-deficient mice (breeder pairs of ENT1-/- mice were kindly provided by Doo-Sup Choi, Mayo Clinic, Rochester, MN). We observed that TGF responses were significantly reduced in ENT1-/- compared to WT mice and that this was independent of genetic background. This observation points to a role of nucleoside transport in the NaCl-synchronous changes of extracellular adenosine levels. Since plasma concentrations of adenosine and of its breakdown product inosine were markedly higher in ENT1-/- than in WT mice one may assume that the direction of adenosine transport in most tissues consists of cellular uptake. Our observations therefore indicate that interference with adenosine disposition by cellular uptake may modify TGF characteristics, perhaps by preventing the translation of alterations of luminal NaCl concentrations into synchronous fluctuations of adenosine levels. An incidental finding of these studies was that spontaneous locomotor activity levels as well as voluntary wheel running activity was significantly reduced in ENT1-/- animals, probably resulting from the central inhibitory effects of the elevated tissue adenosine levels. 3. Angiotensin II has long been known to inhibit renin secretion, an outcome thought to be mediated by angiotensin II receptors (AT1a) on the renin-secreting juxtaglomerular cells and termed short feedback loop inhibition of renin release. Therefore, the explanation for the stimulatory effect of the clinically widely used angiotensin II converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARB) has been escape from short loop feedback inhibition. Using mice with conditional deletion of the Gs alpha gene in renin-producing cells we found that the ACEI captopril and quinaprilate, and the ARB candesartan did not significantly alter plasma renin although these compounds increased plasma renin concentration (PRC) more than 20fold in wild type mice. Since the stimulatory effect of angiotensin II is Gs alpha-dependent, and since angiotensin II receptors are not coupled to Gs alphaz one may conclude that the stimulatory effect of angiotensin II blockade on renin synthesis and release is indirectly mediated by the action of ligand(s) that utilize Gs alpha-dependent pathways. 4. Gap junctions provide a pathway for the functional coupling of individual cells, and such coupling may be important in the communication mechanisms that link the different cells of the juxtaglomerular apparatus. In fact, the gap junctional protein connexin 40 (Cx40) is highly expressed in the extraglomerular mesangium connecting the tubular epithelium at the macula densa and the afferent arteriole. Participation of Cx40 in juxtaglomerular communication is suggested by the finding that the expression of renin is increased and the localization of renin-expressing cells is abnormal in Cx40-deficient mice. Furthermore, in Cx40-deficient mice on two different genetic backgrounds TGF responsiveness was found to be diminished in magnitude and slowed in response velocity suggesting that gap junctional communication may contribute to signal transmission. 5. Fluid reabsorption in tubular segments upstream of the macula densa is part of the JGA-dependent regulation since it determines the NaCl load delivered to the sensing site. We have previously shown that aquaporin 1 in the proximal tubule is required for the high rate of absorption by providing transmembrane entry and exit pathways for water. However, it has remained unclear if water in the proximal tubule can also cross the epithelium through a paracellular pathway. The identification of claudin2 (cldn2) as a tight junction protein highly expressed in renal proximal tubules together with evidence that cldn2 increases water permeability in cultured cells has prompted us to compare proximal fluid reabsorption in wild type and cldn2-deficient mice. We found that cldn2-dependent fluid reabsorption amounted to a fraction of 27.5% of total reabsorption suggesting a sizable paracellular route of proximal fluid transport. Reduced fluid reabsorption in cldn2-deficient mice was associated with a reduced GFR, perhaps a consequence of increased salt delivery to the TGF sensor. We are currently generating mice with deletions of both aquaporin 1 and claudin 2 to examine if the components of fluid reabsorption mediated by membrane or tight junction proteins are independent and additive.
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