Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors

Abstract

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(+/-), and A1AR(-/-) mice. TGF responses to an increase in loop of Henle flow rate from 0 to 30 nl/min, whether determined as change of stop flow pressure or early proximal flow rate, were completely abolished in A1AR(-/-) mice (stop flow pressure response, -6.8 +/- 0.55 mmHg and -0.4 +/- 0.2 in A1AR(+/+) and A1AR(-/-) mice; early proximal flow rate response, -3.4 +/- 0.4 nl/min and +0.02 +/- 0.3 nl/min in A1AR(+/+) and A1AR(-/-) mice). Absence of TGF responses in A1AR-deficient mice suggests that adenosine is a required constituent of the juxtaglomerular signaling pathway. A1AR null mutant mice are a promising tool to study the functional role of A1AR in different target tissues.

Figure 1

(A) Organization of the mouse A1AR gene, the targeting vector, and the mutant allele after recombination. Boxes in black and gray indicate exons and coding regions; relevant restriction sites and location of 5′ and 3′ probes are shown. Herpes simplex virus thymidine kinase (TK) and lacZ were from the pNZTK2 vector (R. D. Palmiter, Howard Hughes Medical Institute, Univ. of Washington, Seattle); the neomycin resistance cassette flanked by two loxP sites (Fl Neo) and driven by the phosphoglycerate kinase promoter were from the Biomedical Core Facilities (Univ. of Michigan, Ann Arbor, MI). (B) Southern blots on HindIII and SacI digests of recombinant ES cell clone DNA showing predicted wild-type and recombinant fragments as indicated by arrows.

Figure 2

(A) RT-PCR of kidney mRNA by using A1AR and β-actin-specific primers in the presence and absence of reverse transcriptase. A1AR RT-PCR products were absent in mRNA from A1AR^−/− mice. (B) Abundance of A1AR mRNA as determined by quantitative RT-PCR (data are means of measurements on three individual RNA samples).

Figure 3

Effect of the A1AR agonist cyclohexyladenosine (CHA) in two different doses (35 and 175 ng) on heart rate (Upper) and systolic blood pressure (Lower) in A1AR^+/+ (n = 10) and A1AR^−/− mice (n = 12).

Figure 4

Average GFR in three successive 15-min clearance periods in six female A1AR^+/+ and nine female A1AR^−/− mice. Dotted lines indicate mean GFR for the 45-min observation period. Vertical lines show SEM.

Figure 5

Recording of stop flow pressure (P_SF, upper traces) and arterial pressure (AP, lower traces) in an A1AR^+/+ and an A1AR^−/− mouse. Black boxes indicate periods of loop of Henle perfusion at 30 nl/min (the gray box represents a flow of 10 nl/min).

Figure 6

Mean stop flow pressure at loop of Henle flow rates of 0 and 30 nl/min in A1AR^+/+, A1AR^+/−, and A1AR^−/− mice. Vertical bars are SEM.

Figure 7

Early proximal flow rate during loop of Henle perfusion at 0 and 30 nl/min in A1AR^+/+ and A1AR^−/− mice. Lines connect measurements in the same tubule. Closed symbols are data from individual nephrons; open symbols are mean values.