Hospital,. Surgery. Depart- ment, 64 Robinson. Street, Waterbury,. CT 06708. ..... Logan,. UT) were performed on all urine and plasma samples. PRA was ..... regional differences in responsiveness of the renal vasculature to adenosine.
Influence Function A. Mariano
AM.
Ibarrola,2
Ibarrola,
partment Medicine, (J. Am.
of Adenosine Receptor and Renal Autoregulation”
E.W.
Edward
Inscho,
of Physiology, New Orleans, Soc.
Nephrol.
P.C. Tulane LA
1991;
W. Inscho,
Van,
L.G.
University
Richard
NaVar,
C. Van,
De-
School
of
Blockade and
L. Gabriel
the hypothesis enhances tubular Key dium
Words: excretion,
Renal
on Renal Navar3
that sodium
endogenous reabsorption
hemodynamics,
adenosine
GFR,
adenosine rate. autoregulation,
so-
receptors
2:991-999)
T
ABSTRACT Experiments were conducted in anesthetized dogs to evaluate the effects of adenosine receptor blockade on renal function and on autoregulation of total RBF and outer cortical blood flow. After control measurements, the adenosine receptor antagonist, I ,3dlpropyl-8-p-sulfophenylxanthine (PSPX) was infused lntrarenally for 45 mm at 2 or 6 M/mln. Responses to PSPX were compared with those obtained during Infusions
of
either
aminophylllne
or
theophylline.
PSPX infusion led to substantial increases in urine flow and sodium excretion (fourto fivefold). RBF increased significantly; however, outer cortical blood flow and GFR were not significantly altered. PRA increased twofold during PSPX infusion. The vasoconstrictor responses to bolus injections of 2-chloroadenoslne (100 mol) were attenuated by 58 and 86% during
the low and
high
doses
of PSPX and
to a lesser
extent with aminophylline/theophylline infusions. At renal arterial pressures above the inflection point, the slope of the average pressure-flow relationship during PSPX infusion was close to zero and was not significantly different from control. Similarly, autoregulatory capability was not altered during infusions of theophylline or aminophylline. These data provide further evidence that endogenous adenosine contributes substantially to the control of renin release but only modestly to the control of RBF and GFR and to renal autoregulatory capability. The natriuretic responses during adenosine blockade, which occurred in the face of elevated renin levels, support
I
Received
2
Current
ment,
April
address
64 Robinson
3 Correspondenceto School of Medicine,
1046-6673/0205-099
15, 1991.
Accepted June 19, 1991. Ibarrola is Waterbury
for Dr. AM. Street,
Waterbury,
Hospital,
Dr. LG. Navar, DepartmentofPhysiology, 1430 Tulane Avenue, New Orleans, 1 $030010
Journal of the American society of Nephrology Copyright C 1991 by the American society of Nephrology
Journal
of the
American
Surgery
Depart-
CT 06708.
Society
of Nephrology
Tulane LA 70112.
University
he nucleotide, adenosine, has been implicated in the regulation of hemodynamics in a variety of organ systems (1). Its hemodynamic actions are complex because most microvascular beds have both A1 and A2 receptor subtypes, which, when activated, can elicit either vasoconstriction (A1) or vasodilation (A2). This biphasic action of exogenous adenosine has created uncertainty over the specific role of endogenous adenosine in the control of renal function (29). Whereas adenosine is considered primarily a vasodilator in many other tissues (1), it has been suggested that adenosine, operating primarily via A1 receptors may exert an important vasoconstrictor influence in the regulation of renal vascular resistance (2-8, 1 0- 1 5). Earlier studies at the whole kidney level suggested that adenosine may be involved in mediating the phenomenon of RBF autoregulation (2,9,16-18); however, Premen et at. (19) were unable to demonstrate that autoregulatory capability was impaired during infusion of aminophylline or high doses of adenosine. In contrast, results from micropuncture experiments were supportive of a role for adenosine in the tubuloglomerular feedback (TGF)mediated changes in afferent arteriolar resistance (6, 1 8). These conclusions were based, to a large cxtent, on results obtained with rather nonspecific agents, such as theophyllmne and isobutylmethylxanthine (IBMX), that may also exert intracellular actions.The recent availability of adenosine receptor blockers that do not exert intracellular actions (20) plus the current resurgence of interest in the possible role of adenosine in renal vascular control mechanisms prompted the study presented here. To the extent that autoregulation at the whole kidney level is a manifestation of the TGF mechanism (2 1), recent results indicating that TGF responses are markedly blunted by the administration of adenosine receptor antagonists (22,23) would predict that adenosine receptor blockade should abolish, or at least attenuate, the capacity of the kidney to autoregulate RBF in response to changes in renal arterial pressure (RAP). Other experiments have also suggested that adenosine may influence sodium excretion by mechanisms independent of changes in filtered load or in
991
Renal
Response
to Adenosine
Blockers
the activity of the renmn-angiotensmn system (2,3,7,9, 1 0, 1 4, 1 5,24). It seemed possible that the more specific adenosine receptor blockers now available (20,22,23) might be able to unmask adenosinerelated components of RBF autoregulation and sodium excretion. In view of previous suggestions that the adenosine vasoconstrictor effects are sustained in the outer nephrons (7,8, 1 3), there was also a possibility that the adenosmne-dependent component of autoregulation is restricted to the outer cortex. Thus, one objective of these experiments was to determine the effects of specific adenosmne receptor blockade on outer cortical blood flow (OCBF) as well as total RBF autoregulatory behavior. Alterations in OCBF autoregulatory responses were evaluated by laser Doppler flowmetry (2 1 ,25). With regard to a possible role for adenosine in regulating sodium excretion, the previous agents used as adenosine receptor antagonists could have exerted other intracellular effects, possibly confounding the data obtained. Therefore, the influence of endogenous adenosmne on whole kidney sodium excretion was evaluated before and after induction of adenosine receptor blockade when a more specific extracellular receptor antagonist 1 ,3dipropyl-8-p-sulfophenylxanthmne (PSPX) was used.
METHODS Animal
Preparation
Experiments were performed on 1 5 adult mongrel dogs weighing between 1 4 and 24 kg that had been maintained on a standard dog chow. The dogs were fasted for 1 6 to 20 hours before the experimental day. The animals were anesthetized with sodium pentobarbital (25 mg/kg i.v.) and were prepared for autoregulation and renal clearance studies as previously described (2 1 ). A tracheostomy was performed, and respiration was regulated mechanically (Harvard Apparatus, South Natick, MA). A catheter was placed in the left jugular vein for infusion of inulin at a rate sufficient to maintain a plasma concentration of approximately 0.2 mg/mL. Arterial pressure was measured through a catheter inserted into the femoral artery, which was connected to a Statham pressure transducer (Statham Laboratories, Hato Rey, PR). Blood samples were also collected through this catheter. The left kidney was exposed through a flank incision, and the proximal renal artery and the ureter were dissected free of adjacent tissue. An electromagnetic flow transducer was placed around the renal artery and connected to a square-wave flowmeter (Carolina Medical Electronics, King, NC) to measure RBF. Flow and pressure data were recorded on a Grass Polygraph (Grass Instruments, Qumncy, MA). The ureter was catheterized to allow timed urine collections. Autoregulation curves were generated in response
992
to graded reductions in RAP with an adjustable plastic clamp placed around the renal artery distal to the flow probe. RAP was measured through a curved 23gauge needle inserted in the renal artery distal to the plastic clamp and connected to a Statham pressure transducer. The renal arterial line was infused with 0.4 mL/min of hepaninized, isotonic saline solution to maintain patency. As previously described (21), a small area ( 1 cm2) of the renal capsule was carefully removed and a laser Doppler flow probe (MedPacific, Seattle, WA) was oriented over the outer cortical surface of the kidney. The voltage output of the unit is proportional to blood flow velocity in the microvasculature of approximately 1 mm3 of tissue directly under the probe (25). Output was recorded on a Grass Polygraph, and flow was expressed as millimeters of pen deflection (units) for comparison purposes. Zero flow was determined as the signal generated while the renal artery was completely occluded.
Experimental
Protocols
Forty-five minutes were allowed between completion of surgery and initiation of the experimental procedures. During each experiment, mean arterial pressure, RAP, OCBF, and RBF were continuously monitored. Two or three timed urine collections of 15 to 30 mm each were made during each experimental period for determination of average inulin clearance (GFR), urine flow, and sodium excretion. A blood sample was collected at the midpoint of each timed urine collection. Microhematocrit measurements were performed on all arterial blood samples. At the end of the surgical equilibration period, an arterial blood sample was collected for the measurement of control PRA. Control autoregulation curves for RBF and OCBF were obtained by adjusting the arterial clamp to produce stepwise reductions in RAP of 10 to 20 mm Hg. Renal function was allowed to stabilize for 2 to 3 mm after each reduction in pressure. After the last reduction in RAP, the clamp was released to reestablish control RAP. Once renal function had stabilized, the RBF responses to intra-anterial 1 0-s bolus injections (1 0 and 1 00 tmol) of the adenosine agonist, 2-chloroadenosine (2-CA), were observed. After the effects of these test doses had waned, control renal clearances and an arterial blood sample were obtained. After control measurements, an adenosine receptor antagonist, PSPX (1 ,3,dipropyl-8-p-sulfophenylxanthmne), at 2 or 6 imol/min (N = 9 and 6, respectively) was infused into the renal artery for 45 mm. PSPX concentration in the infusion solution was adjusted to allow volume infusion rate into the renal artery to remain at 0.4 mL/min. Renal clearance periods and a blood sample for PRA were again obtained. After the clearance periods, RAP was reduced to generate autoregulation
Volume
2
-
Number
5
‘
1991
lbarrola
curves for RBF and OCBF. After recovery of RBF, the efficacy of adenosine receptor antagonism was determined by comparing the RBF responses during PSPX infusion to identical bolus injections of 2-CA as before. The PSPX infusion was discontinued, and the above protocol was repeated during infusion with ammnophyllmne or theophyllmne at 1 0 (N = 5) or 20 (N = 6) mol/min in order to determine if theophylline or ammnophyllmne exerted effects different from or in addition to those observed during PSPX treatment and to compare the present results with those previously reported with these agents (1 7, 1 9). Because ammnophyllmne is simply a soluble salt of theophylline plus the relatively inert ethylenediammne, the data from those two groups were combined and expressed as ammno/theo. At the end of each experiment, the electromagnetic flow probe was calibrated in situ by the placement of a catheter directly into the renal artery and collection of timed blood samples into a graduated cylinder. The kidney was excised, stripped of surrounding tissue and capsule, blotted dry, and weighed.
Analyses
and
Calculations
Inulin concentration in both urine and plasma samples were measured by an anthrone colorimetric technique, and GFR was calculated by the standard clearance formula. RPF was derived from the measured values for RBF and microhematocnit. Measurements of sodium and potassium concentrations (Instrumentation Laboratory, Lexington, MA) and osmolality (Wescor, Logan, UT) were performed on all urine and plasma samples. PRA was measured by a commercially available kit (GammaCoatC [‘25IJPlasma Renin Activity; Dade Baxter Travenol Diagnostics, Inc. Cambridge, MA) adapted from the method of Haber et at. (26) and designed for the quantitative determination of PRA in human plasma by the RIA of generated angiotensmn I. The sensitivity of the assay was calculated from the 95% confidence limits of the zero angiotensmn I standard and was 0.018 ng of angiotensin I/i 00 AL (three assays). The percent binding of the angiotensmn I antibody was 56 ± 2% with a nonspecific binding of 1 .7 ± 0.3%. The intraassay and mnterassay (N = 3) coefficients of variations were 3 and 7%, respectively. The compounds used in the current study (2-CA, PSPX, ammnophyllmne, and theophyllmne) were purchased from Research Biochemicals Inc. (Natick, MA). All drugs were dissolved in 0.9% NaC1 solution and were freshly prepared on the day of each experiment. ,
Statistical Values panisons
Journal
Analysis are reported of quantitative
of the
American
as mean ± SE. Statistical data were made by the
Society
of Nephrology
compaired
et al
t test or by analysis of variance with repeated measures in conjunction with the Newman-Keuls multiple range test where appropriate. A P value
