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【摘要】 Adjustments of renal vascular resistance in response to changes in blood pressure are mediated by an interplay between the myocyte-inherent myogenic and the kidney-specific tubuloglomerular feedback (TGF) mechanisms. Using mice with deletion of the A 1 adenosine receptor (A1AR) gene, we tested the prediction that the absence of TGF, previously established to result from A1AR deficiency, is associated with a reduction in the efficiency of autoregulation. In anesthetized wild-type (A1AR+/+) and A1AR-deficient mice (A1AR-/-), glomerular filtration rate (GFR) and renal blood flow (RBF) were determined before and after reducing renal perfusion pressure through a suprarenal aortic clamp. In response to a blood pressure reduction by 15.9 ± 1.34 mmHg in A1AR-/- ( n = 9) and by 14.2 ± 0.9 mmHg in A1AR+/+ mice ( n = 8; P = 0.31), GFR fell by 187.9 ± 37 µl/min and by 72.3 ± 10 µl/min in A1AR-/- and A1AR+/+ mice, respectively ( P = 0.013). Similarly, with pressure reductions of 14.8 ± 1.1 and 13.3 ± 1.5 mmHg in A1AR-/- ( n = 9) and wild-type mice ( n = 8), respectively ( P = 0.43), RBF fell by 0.17 ± 0.02 ml/min in A1AR-/- mice and by only 0.08 ± 0.02 ml/min in wild-type animals ( P = 0.0039). Autoregulatory indexes for both GFR and RBF were significantly higher in A1AR-/- compared with A1AR+/+ mice, indicating reduced regulatory responsiveness in the knockout animals. We conclude that autoregulation of renal vascular resistance is less complete in A1AR-deficient mice, an effect that is presumably related to absence of TGF regulation in these animals.
【关键词】 glomerular filtration rate renal blood flow aortic clamp autoregulatory index
CHANGES IN ARTERIAL BLOOD pressure induce adjustments in renal vascular resistance that result in near constancy of renal blood flow (RBF) and glomerular filtration rate (GFR), a phenomenon generally referred to as autoregulation. Substantial experimental and modeling evidence supports the concept that autoregulation of renal vascular resistance is caused by an interplay between at least two and perhaps more than two different mechanisms ( 8 ). The myogenic mechanism reflects an inherent property of most vascular smooth muscle cells to constrict in response to mechanical signals generated by increased stretch or wall tension. The tubuloglomerular feedback (TGF) mechanism, on the other hand, is a specifically renal regulatory system that is activated by changes in distal tubule NaCl concentration that result from pressure-dependent alterations in GFR and tubular reabsorption ( 16 ).
Recent studies addressing the question of the extracellular mediator of TGF have shown that TGF responses are absent in mice with targeted deletion of the A 1 adenosine receptor (A1AR) ( 2, 19 ). Because TGF is believed to be responsible, in part, for renal autoregulation as indicated above, one would expect that the precision of pressure-dependent regulation of renal resistance may be impaired in A1AR-deficient mice. On the other hand, it is conceivable that in the chronic absence of TGF other autoregulatory mechanisms may be able to compensate and produce the resistance changes required for maintenance of RBF and GFR.
The present series of experiments was performed to examine the steady-state response of RBF and GFR to an acute step change in arterial pressure in wild-type and A1AR-deficient mice. Our results show that the effectiveness of autoregulation of both RBF and GFR is significantly diminished in A1AR-/- compared with wild-type mice.
METHODS
Experiments were performed in mice of the A1AR strain generated in this laboratory ( 19 ). Brother-sister mating of heterozygous mice generated wild-type and -/- genotypes in a mixed 129J/C57BL6 genetic background. Genotyping was done on tail DNA using PCR as described previously ( 19 ). Mice were kept on standard rodent chow and tap water. Animal care and experimentation were approved and carried out in accordance with institutional and National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Mice were anesthetized with 100 mg/kg inactin intraperitoneally and 100 mg/kg ketamine intramuscularly. Cannulas were placed in the trachea, in the femoral artery for measurement of arterial blood pressure, and in the jugular vein for an intravenous maintenance infusion of 2.25 g/dl BSA in saline at a rate of 0.35 ml/h. Through a flank incision, an adjustable clamp was placed around the aorta proximal to the origin of the two renal arteries, and it was held in place by fixing it to a micromanipulator.
GFR. In addition to the vascular catheters, the bladder was catheterized through a suprapubic incision. To determine GFR, mice were infused with 125 I iothalamate (Glofil, Questcor Pharmaceutical, Hayward, CA) at 5 µCi/h. After 30-45 min of equilibration, a blood sample of 4 µl was collected into heparinized 5-µl microcaps (Drummond, Broomall, PA), and three consecutive 10-min urine collection periods were made. After a second blood collection, the lower body blood pressure was reduced by tightening the aortic clamp. After a 10-min waiting period, three additional 10-min urine collections were made followed by a terminal blood collection. 125 I iothalamate radioactivity was measured in duplicate 0.5-µl aliquots of plasma and urine in a Riastar counter (Packard Instrument, Downers Grove, IL). Urine volume was determined gravimetrically. The autoregulatory index (AI) was calculated from (GFR2 - GFR1)/GFR1 divided by (MAP2 - MAP1)/MAP1 where GFR1 and MAP1 are GFR and mean arterial pressure (MAP) at the high pressure, and GFR2 and MAP2 are GFR and MAP at the reduced pressure.
Measurements of total RBF. The left renal artery was approached from a flank incision and carefully dissected free to permit placement of a 0.5-mm V-type ultrasonic flow probe (Transonic Systems, Ithaca, NY). The probe was held in place with a micromanipulator. The size of the probe available for these studies demanded the use of mice with body weights greater than 25 g. RBF signals were digitized and analyzed using PowerLab software ( ADInstruments, Colorado Springs, CO). After stability was achieved, RBF was recorded for 10 min. Subsequently, the aortic clamp was tightened to achieve an 15-mmHg reduction in femoral artery pressure, and RBF was recorded for another 10 min. AI was calculated as indicated above with RBF replacing GFR.
Statistics. Comparisons between wild-type and A1AR-/- mice were done by unpaired t -test. For comparisons within groups, we used the paired t -test or ANOVA with repeated measures and Bonferroni correction.
RESULTS
GFR. The effect of a blood pressure reduction on GFR was examined in eight wild-type and nine A1AR-/- mice. Mean body and kidney weights were similar between genotypes (31.2 ± 1.8 g and 361 ± 18 mg in wild-type, and 31 ± 1.6 g and 360 ± 28 mg in A1AR-/- mice). The time course of changes in mean femoral blood pressure and GFR in response to aortic clamping is shown in Fig. 1 A. There was no significant difference in femoral artery pressure under baseline conditions between anesthetized wild-type and A1AR-/-, and aortic clamping reduced blood pressure to the same extent in both genotypes. There was also no significant difference between GFR of wild-type and A1AR-/- mice during the control periods, but GFR fell significantly more in response to aortic clamping in A1AR-/- than wild-type mice. In fact, when tested by repeated-measures ANOVA, none of the experimental GFR measurements was different from any of the control values in wild-type mice. In contrast, in A1AR-/- mice all measurements during reduced arterial pressure were significantly different from all control values. Thus GFR of A1AR-/- at the reduced pressure was lower in two of the experimental periods compared with wild-type (unpaired t -test). Mean changes in arterial pressure and GFR for all three periods are shown in Fig. 2. While the mean decrease in arterial pressure was not different between wild-type and A1AR-/- mice, mean GFR decreased significantly more in A1AR-/- than wild-type mice (187.9 ± 37 vs. 72.3 ± 10 µl/min; P = 0.013). Autoregulatory indexes of individual experiments are shown in Fig. 3, left. Mean AI was 1.15 ± 0.2 in wild-type and 2.47 ± 0.37 in A1AR-/- mice ( P = 0.009). Urine flows of wild-type mice averaged 1.9 ± 0.2 µl/min in control and 1.4 ± 0.2 µl/min after pressure reduction ( P = 0.0007), whereas in A1AR-/- urine flow fell from 1.95 ± 0.2 to 1.25 ± 0.15 µl/min with pressure reduction ( P = 0.0016). Time control experiments have shown that in the absence of an intervention, GFR and arterial pressure are stable over a 1-h observation period in both wild-type and A1AR-deficient mice ( Fig. 1 B ).
Fig. 1. A : mean femoral artery blood pressure ( left ) and glomerular filtration rate (GFR; right ) in wild-type (A1AR+/+; n = 8) and A1AR-/- mice ( n = 9) before and after suprarenal aortic constriction at the 30-min time point. GFR values are means for 6 successive clearance measurements plotted in the middle of the urine collection period. Vertical bars are SE. Numbers indicate P values for comparisons between A1AR+/+ and A1AR-/- in a given clearance period. B : time control experiments in which mean femoral artery blood pressure ( left ) and GFR ( right ) were measured in wild-type (A1AR+/+; n = 8) and A1AR-/- mice ( n = 8) over a 1-h time period. * P < 0.05, ** P < 0.01 (repeated-measures ANOVA).
Fig. 2. Mean reductions in femoral artery blood pressure ( left ) and GFR ( right ) in A1AR+/+ and A1AR-/- mice.
Fig. 3. Autoregulation indexes of GFR ( left ) and renal blood flow (RBF; right ) in individual experiments. Horizontal lines indicate mean values.
RBF. The effect of a blood pressure reduction on RBF was tested in eight wild-type and nine A1AR-/- mice. Mean body and kidney weights were 35 ± 3 g and 208 ± 22 mg in wild-type and 35 ± 1.3 g and 227 ± 13 mg in A1AR-/- mice. Mean femoral blood pressure during control was 98 ± 3.1 mmHg in wild-type and 96 ± 2.4 mmHg in A1AR-/- mice. Aortic clamping reduced femoral pressure to 84 ± 3 mmHg in wild-type and to 80 ± 3 mmHg in A1AR-/- mice. RBF during control averaged 1.2 ± 0.1 ml/min in wild-type and 1.44 ± 0.2 ml/min in A1AR-/- mice ( P = 0.44 vs. wild type). RBF fell in response to the pressure reduction to 1.12 ± 0.1 ml/min or by 6.7 ± 1.2% in wild-type ( P = 0.001 vs. control), and to 1.27 ± 0.1 or by 12 ± 0.7% in A1AR-/- mice ( P < 0.0001 vs. control). The changes in arterial pressure, RBF, and renal vascular resistance (RVR) are shown in Fig. 4. While arterial pressure decreased to the same extent in wild-type and A1AR-/- (by 13.3 ± 1.5 and 14.8 ± 1.1 mmHg; P = 0.43), RBF decreased by 0.08 ± 0.02 ml/min in wild-type and by 0.17 ± 0.02 ml/min in A1AR-/- mice ( P = 0.0039). Accordingly, RVR fell significantly more in wild-type than A1AR-/- mice (by 6.7 ± 1.7 and 2.5 ± 0.8 mmHg·min -1 ·ml -1, respectively; P = 0.025). Individual values of the AI are shown in Fig. 3, right. On average, AI increased from 0.49 ± 0.07 in wild-type to 0.79 ± 0.05 in A1AR-/- ( P = 0.002).
Fig. 4. Mean reductions in femoral artery blood pressure ( left ), RBF ( middle ), and renal vascular resistance (RVR; right ) in A1AR+/+ and A1AR-/- mice. SE values are indicated by vertical bars.
DISCUSSION
The present study shows that the adjustment of RVR to a reduction in arterial pressure is less effective in A1AR-deficient mice than in normal animals. As a consequence, both RBF and GFR decrease to a greater extent in mutant than wild-type mice. Taken at face value, this observation indicates that the vasorelaxation characteristically elicited by a fall in blood pressure is significantly diminished when A1AR are nonfunctional. This finding implies that the reduction in RVR induced by a blood pressure decrease is caused, in part, by removal of an A1AR-dependent vasoconstrictor tone. As a first step to explore the role of A1AR in autoregulation, we tested the autoregulatory ability by determining the steady-state response of GFR and RBF to a step reduction in arterial pressure. The assessment of steady-state autoregulation has remained a valid approach to evaluate basic mechanisms of pressure-dependent resistance adjustments ( 1 ). While the analysis of the dynamic characteristics of autoregulation has yielded important new insights into pressure-dependent resistance changes ( 3 ), the use of this approach in mice represents a formidable experimental challenge that will require further technical advances.
The cause for the impaired autoregulation in A1AR-/- mice is likely to be related to their inability to regulate the arteriolar tone through the TGF mechanism ( 2, 19 ). Impairment of TGF by various means has been previously shown to affect the precision of pressure-dependent resistance adjustments. Pressure dependency of SNGFR was noted regardless of whether the TGF loop was physically disrupted by injecting an oil block, blocked acutely by adding furosemide to the perfusate ( 8 ), or inhibited by chronic treatment with DOCA and a high-salt diet ( 8 ). In contrast, arterial pressure had little effect on GFR when the TGF loop was intact ( 6, 8, 9, 12, 13, 17 ). In the in vitro-perfused juxtamedullary nephron preparation, interference with the TGF mechanism by furosemide or physical interruption of the feedback loop markedly diminished autoregulatory diameter alterations of afferent arterioles ( 7, 15, 20 ), and constancy of afferent arteriolar blood flow was no longer maintained ( 20 ).
Recently, enhancement of pressure-dependent dilatation by a reduction in extracellular adenosine has been observed in the isolated hydronephrotic kidney preparation ( 21 ). Because this preparation is devoid of TGF, the authors concluded that adenosine inhibits myogenic vasoconstriction by activating high-affinity A2aAR. To the extent that the postulate of a reduction of adenosine levels around the afferent arteriole during reduced arterial pressure is correct, this mechanism would in itself enhance, not diminish, low pressure-induced dilatation. Whether the long-standing A1AR deficiency of knockout mice may affect the myogenic component of autoregulation in some other way remains to be determined.
A role of adenosine in autoregulation has been investigated previously by assessing the effects of blood pressure changes on GFR and/or RBF following the administration of methylxanthines. Although the results are not entirely homogeneous ( 11 ), several more recent studies have reported well-maintained autoregulation in the presence of aminophylline, 1,3-dipropyl-8-p-sulfophenylxanthine (PSPX), or 3-isobutyl-1-methylxanthine (IBMX) ( 4, 10, 14 ). Because in these studies the adenosine receptor blockers were given acutely, it is possible that differences in the duration of the blockade are responsible for the different response of autoregulation to adenosine receptor blockade. On the other hand, it is unknown whether complete inhibition of A1AR in association with full TGF blockade was achieved in these studies. In addition, aminophylline, PSPX, and IBMX are nonspecific blockers of adenosine receptors, raising the possibility that simultaneous inhibition of all adenosine receptors produces results that differ from those achieved by more selective approaches. Finally, the specificity of methylxanthines as inhibitors of adenosine receptors is not perfect. Especially their well-established role as inhibitors of phosphodiesterases may complicate data interpretation ( 18 ). More recently, studies in the juxtamedullary nephron preparation have shown that the perfusion pressure-induced constriction of afferent arterioles was not significantly altered by the A1AR-specific antagonist 1,3-dipropyl-8-cyclopentylxanthine ( 5 ). This finding suggests that A1AR may not play a dominant role in the autoregulation of juxtamedullary vessels, although absence of TGF in juxtamedullary nephrons of A1AR-/- mice has not yet been established. Nevertheless, it is conceivable that the mechanisms underlying autoregulation may quantitatively or qualitatively differ between surface and deep nephrons.
In summary, the absence of functional a A1AR in mice is associated with a reduced ability to maintain constancy of GFR and RBF during changes of arterial blood pressure. In view of the previously demonstrated lack of TGF regulation in A1AR-/- mice, it is likely that the impaired autoregulatory effectiveness reflects the absence of the TGF-dependent component of renal autoregulation.
GRANTS
This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health. S. Hashimoto was the recipient of a Visiting Fellowship of NIDDK.
【参考文献】
Bidani AK, Hacioglu R, Abu-Amarah I, Williamson GA, Loutzenhiser R, and Griffin KA. "Step" vs. "dynamic" autoregulation: implications for susceptibility to hypertensive injury. Am J Physiol Renal Physiol 285: F113-F120, 2003.
Brown R, Ollerstam A, Johansson B, Skott O, Gebre-Medhin S, Fredholm B, and Persson AE. Abolished tubuloglomerular feedback and increased plasma renin in adenosine A1 receptor-deficient mice. Am J Physiol Regul Integr Comp Physiol 281: R1362-R1367, 2001.
Holstein-Rathlou NH and Marsh DJ. Renal blood flow regulation and arterial pressure fluctuations: a case study in nonlinear dynamics. Physiol Rev 74: 637-681, 1994.
Ibarrola AM, Inscho EW, Vari RC, and Navar LG. Influence of adenosine receptor blockade on renal function and renal autoregulation. J Am Soc Nephrol 2: 991-999, 1991.
Inscho EW, Cook AK, Imig JD, Vial C, and Evans RJ. Physiological role for P2X1 receptors in renal microvascular autoregulatory behavior. J Clin Invest 112: 1895-1905, 2003.
Moore LC. Tubuloglomerular feedback and SNGFR autoregulation in the rat. Am J Physiol Renal Fluid Electrolyte Physiol 247: F267-F276, 1984.
Moore LC and Casellas D. Tubuloglomerular feedback dependence of autoregulation in rat juxtamedullary afferent arterioles. Kidney Int 37: 1402-1408, 1990.
Moore LC, Schnermann J, and Yarimizu S. Feedback mediation of SNGFR autoregulation in hydropenic and DOCA- and salt-loaded rats. Am J Physiol Renal Fluid Electrolyte Physiol 237: F63-F74, 1979.
Navar LG, Burke TJ, Robinson RR, and Clapp JR. Distal tubular feedback in the autoregulation of single nephron glomerular filtration rate. J Clin Invest 53: 516-525, 1974.
Ogawa N and Ono H. Modulation of cyclic AMP and autoregulation of renal blood flow, analysed by the use of forskolin and 1-methyl-3-isobutylxanthine. J Pharm Pharmacol 40: 207-209, 1988.
Ono H, Inagaki K, and Hashimoto K. A pharmacological approach to the nature of the autoregulation of the renal blood flow. Jpn J Physiol 16: 625-634, 1966.
Ploth DW, Dahlheim H, Schmidmeier E, Hermle M, and Schnermann J. Tubuloglomerular feedback and autoregulation of glomerular filtration rate in Wistar-Kyoto spontaneously hypertensive rats. Pflügers Arch 375: 261-267, 1978.
Ploth DW, Schnermann J, Dahlheim H, Hermle M, and Schmidmeier E. Autoregulation and tubuloglomerular feedback in normotensive and hypertensive rats. Kidney Int 12: 253-267, 1977.
Premen AJ, Hall JE, Mizelle HL, and Cornell JE. Maintenance of renal autoregulation during infusion of aminophylline or adenosine. Am J Physiol Renal Fluid Electrolyte Physiol 248: F366-F373, 1985.
Sanchez-Ferrer CF, Roman RJ, and Harder DR. Pressure-dependent contraction of rat juxtamedullary afferent arterioles. Circ Res 64: 790-798, 1989.
Schnermann J and Briggs JP. Function of the juxtaglomerular apparatus: control of glomerular hemodynamics and renin secretion. In: The Kidney Physiology and Pathophysiology, edited by Seldin DW and Giebisch G. Philadelphia, PA: Lippincott Williams and Wilkins, 2000, p. 945-980.
Schnermann J, Briggs JP, and Weber PC. Tubuloglomerular feedback, prostaglandins, and angiotensin in the autoregulation of glomerular filtration rate. Kidney Int 25: 53-64, 1984.
Smellie FW, Davis CW, Daly JW, and Wells JN. Alkylxanthines: inhibition of adenosine-elicited accumulation of cyclic AMP in brain slices and of brain phosphodiesterase activity. Life Sci 24: 2475-2482, 1979.
Sun D, Samuelson LC, Yang T, Huang Y, Paliege A, Saunders T, Briggs J, and Schnermann J. Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors. Proc Natl Acad Sci USA 98: 9983-9988, 2001.
Takenaka T, Harrison-Bernard LM, Inscho EW, Carmines PK, and Navar LG. Autoregulation of afferent arteriolar blood flow in juxtamedullary nephrons. Am J Physiol Renal Fluid Electrolyte Physiol 267: F879-F887, 1994.
Tang L, Parker M, Fei Q, and Loutzenhiser R. Afferent arteriolar adenosine A2a receptors are coupled to KATP in in vitro perfused hydronephrotic rat kidney. Am J Physiol Renal Physiol 277: F926-F933, 1999.
作者单位：National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland