Adenosine is a determinant of metabolic control of organ function increasing oxygen supply through the A2 class of adenosine receptors and reducing oxygen demand through A1 adenosine receptors (A1AR). In the kidney, activation of A1AR in afferent glomerular arterioles has been suggested to contribute to tubuloglomerular feedback (TGF), the vasoconstriction elicited by elevations in [NaCl] in the macula densa region of the nephron. To further elucidate the role of A1AR in TGF, we have generated mice in which the entire A1AR coding sequence was deleted by homologous recombination. Homozygous A1AR mutants that do not express A1AR mRNA transcripts and do not respond to A1AR agonists are viable and without gross anatomical abnormalities. Plasma and urinary electrolytes were not different between genotypes. Likewise, arterial blood pressure, heart rates, and glomerular filtration rates were indistinguishable between A1AR ؉/؉ , A1AR

Experiments were performed to examine the effect of changes in dietary salt intake on the neuronal form of the constitutive nitric oxide synthase (ncNOS, type I NOS), renin, and angiotensinogen mRNA expression in the kidney. Three groups of Sprague-Dawley rats were studied as follows: rats maintained on a 3% Na diet plus 0.45% NaCl in the drinking fluid for 7 days (high salt), rats given a single injection of furosemide (2 mg/kg i.p.) and a 0.03% Na diet for 7 days (low salt), and rats on a diet containing 0.2% Na (control). mRNA expression was assessed with reverse transcription-polymerase chain reaction (RT-PCR) methods using cDNA prepared from samples of renal cortex and microdissected tubular segments. ncNOS PCR products were quantified by comparison with a dilution series of a mutant deletion template. Compared with their respective control, ncNOS mRNA levels in renal cortical tissue were elevated in rats on a low-salt diet and reduced in rats on a high-salt diet. Similar changes were seen in the expression of renin and angiotensinogen mRNA. Dietary salt intake did not alter the mRNA levels for ncNOS from the inner medulla or for endothelial constitutive NOS (ecNOS, type III NOS) and inducible NOS (iNOS, type II NOS) in the renal cortex. ncNOS mRNA was found in glomeruli dissected with the macula densa-containing segment (MDCS), but only at marginal levels in glomeruli without MDCS. Furthermore, a low-salt diet stimulated ncNOS mRNA in glomeruli with MDCS by 6.2-fold compared with a high-salt diet. There was no effect of salt diet on ncNOS mRNA in glomeruli without MDCS or in inner medullary collecting ducts. These results suggest that ncNOS expression in macula densa cells is inversely regulated by salt intake, thus following the known response of the renin-angiotensin system to changes in salt balance.

Transport inhibitor and ion substitution studies were performed using perfused, superfused preparations of the isolated rabbit juxtaglomerular apparatus to investigate transport dependency of macula densa-mediated renin secretion. In the first experimental series, tubular perfusion with a high-NaCl solution containing 10(-6) M bumetanide increased renin secretion compared with perfusion with high NaCl alone from 8.7 to 24.6 nano-Goldblatt hog units (nGU)/min. Bath addition of 10(-6) M bumetanide had no effect on renin release. The second series tested ability of luminal addition of 54 mmol/l Na or Cl salts to inhibit renin secretion, starting from a stimulated value produced by low-NaCl perfusion. Perfusion with a high-NaCl solution decreased renin secretion from 58.9 to 14.8 nGU/min, which served as a positive control. Addition of choline chloride decreased renin secretion from 42.7 to 16.6 nGU/min, and RbCl decreased renin secretion from 54.9 to 17.0 nGU/min. In contrast, addition of two different Na salts had no effect on renin release (from 41.7 to 31.6 nGU/min with sodium isethionate and from 14.1 to 13.5 nGU/min with sodium acetate). Also, in the presence of 26 mmol/l Cl, addition of 54 mmol/l Na had no effect on renin secretion (29.9-36.8 nGU/min). These data demonstrate that renin secretion is directly stimulated by luminal application of transport blockers and can be inhibited by increases in Cl concentration at the macula densa but not by changes in Na concentration. These results support the hypothesis that the initiating signal for macula densa control of renin secretion is an inverse change in transport rate via the luminal Na(+)-K(+)-2Cl- cotransporter.

Three different full-length splice isoforms of the Na-K-2Cl co-transporter (NKCC2/BSC1) are expressed along the thick ascending limb of Henle (TAL), designated NKCC2A, NKCC2B, and NKCC2F. NKCC2F is expressed in the medullary, NKCC2B mainly in the cortical, and NKCC2A in medullary and cortical portions of the TAL. NKCC2B and NKCC2A were shown to be coexpressed in the macula densa (MD) segment of the mouse TAL. The functional consequences of the existence of three different isoforms of NKCC2 are unclear. For studying the specific role of NKCC2A in kidney function, NKCC2A؊/؊ mice were generated by homologous recombination. NKCC2A؊/؊ mice were viable and showed no gross abnormalities. Ambient urine osmolarity was reduced significantly in NKCC2A؊/؊ compared with wild-type mice, but water deprivation elevated urine osmolarity to similar levels in both genotypes. Baseline plasma renin concentration and the effects of a high-and a low-salt diet on plasma renin concentration were similar in NKCC2A؉/؉ and ؊/؊ mice. However, suppression of renin secretion by acute intravenous saline loading (5% of body weight), a measure of MD-dependent inhibition of renin secretion, was reduced markedly in NKCC2A؊/؊ mice compared with wild-type mice. Cl and water absorption along microperfused loops of Henle of NKCC2A؊/؊ mice were unchanged at normal flow rates but significantly reduced at supranormal flow. Tubuloglomerular feedback function curve as determined by stop flow pressure measurements was left-shifted in NKCC2A؊/؊ compared with wild-type mice, with maximum responses being significantly diminished. In summary, NKCC2A activity seems to be required for MD salt sensing in the high Cl concentration range. Coexpression of both high-and low-affinity isoforms of NKCC2 may permit transport and Cl-dependent tubuloglomerular feedback regulation to occur over a wider Cl concentration range. T he Na-K-2Cl co-transporter (NKCC2), located in the apical membrane of the epithelial cells of the thick ascending limb of Henle (TAL), constitutes the major cellular uptake pathway in this portion of the nephron. In addition, NKCC2 transport activity in macula densa (MD) cells is considered to be the initial step in the signaling chain that links tubular epithelial cells of the TAL with vascular cells of the afferent arteriole (1-5). At least four different full-length splice isoforms of NKCC2 are expressed along the TAL, designated NKCC2A, NKCC2B, NKCC2F, and NKCC2AF (6 -10). These isoforms of the transporter are derived from differential splicing of the variable exon 4 of the NKCC2 gene (7,8,11). Exon 4 encodes for amino acids of the second transmembrane domain and the adjacent intracellular loop of NKCC2, a region that has been shown to be involved in the ion-binding characteristics of the co-transporter (11). Cell-specific splicing results in heterogeneous expression of the various NKCC2 isoforms along the TAL. NKCC2F, the most abundant NKCC2 isoform, is expressed in the medullary TAL, NKCC2B mainly in the cortical TAL, and NKCC2A both in medullar...

Abstract. Adenosine induces vasoconstriction of renal afferent arterioles through activation of A1 adenosine receptors (A1AR). A1AR are directly coupled to Gi/Go, resulting in inhibition of adenylate cyclase, but the contribution of this signaling pathway to smooth muscle cell activation is unclear. In perfused afferent arterioles from mouse kidney, adenosine and the A1 agonist N 6