Platelet-activating factor and solute transport processes in
the kidney
Rajash K.
Handa,
Jack W.
Strandhoy,
Carlos
E.
Giammattei, and
Shelly E.
Handa
Department of Physiology and Pharmacology, Wake Forest
University School of Medicine, Winston-Salem, North Carolina 27157
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ABSTRACT |
We examined the
hemodynamic and tubular transport mechanisms by which
platelet-activating factor (PAF) regulates salt and water excretion. In
anesthetized, renally denervated male Wistar rats, with raised systemic
blood pressure and renal arterial blood pressure maintained at normal
levels, intrarenal PAF infusion at 2.5 ng · min
1 · kg
1
resulted in a small fall in systemic blood pressure (no change in renal
arterial blood pressure) and an increase in renal blood flow and
urinary water, sodium, and potassium excretion rates. The PAF-induced
changes in cardiovascular and renal hemodynamic function were abolished
and renal excretory function greatly attenuated by treating rats with a
nitric oxide synthase inhibitor. To determine whether a tubular site of
action was involved in the natriuretic effect of PAF, cortical proximal
tubules were enzymatically dissociated from male Wistar rat kidneys,
and oxygen consumption rates (QO2) were used as
an integrated index of transcellular sodium transport. PAF at 1 nM
maximally inhibited QO2 in both untreated and
nystatin-stimulated (sodium entry into renal cell is not rate limiting)
proximal tubules by ~20%. Blockade of PAF receptors or
Na+-K+-ATPase pump activity with BN-52021 or
ouabain, respectively, abolished the effect of PAF on
nystatin-stimulated proximal tubule QO2.
Inhibition of nitric oxide synthase or guanylate cyclase systems did
not alter PAF-mediated inhibition of nystatin-stimulated proximal
tubule QO2, whereas phospholipase
A2 or cytochrome-P-450 monooxygenase inhibition
resulted in a 40-60% reduction. These findings suggest that
stimulation of PAF receptors on the proximal tubule decreases
transcellular sodium transport by activating phospholipase
A2 and the cytochrome-P-450 monooxygenase
pathways that lead to the inhibition of an ouabain-sensitive component of the basolateral Na+-K+-ATPase pump. Thus PAF
can activate both an arachidonate pathway-mediated suppression of
proximal tubule sodium transport and a nitric oxide pathway-mediated
dilatory action on renal hemodynamics that likely contributes to the
natriuresis and diuresis observed in vivo.
nitric oxide; vasopressin; blood pressure; renal blood flow; urinary water; electrolyte excretion; guanylate cyclase; phospholipase
A2; cytochrome P-450 monooxygenase; sodium-potassium-adenosine 5'-triphosphatase; proximal tubule; oxygen
consumption; rat
 |
INTRODUCTION |
PLATELET-ACTIVATING FACTOR (PAF)
represents a group of
1-alkyl-2-acetyl-sn-glycero-3-phosphocholines that is a
class of lipid mediators involved in many physiological (e.g.,
cell-cell signaling, regulation of blood pressure, and reproduction
biology) and pathological (e.g., endotoxic/septic shock, immunological
and inflammatory diseases, and ischemia and reperfusion injury)
processes in the body (15, 28). Much of the research on
PAF and the kidney has focused on the role of PAF in renal vascular and
glomerular function. This is understandable given that PAF receptor
antagonists can improve the outcome of many kidney diseases with a
vascular or glomerular etiology (22, 31). Consequently,
little is known about the possible actions of PAF on other aspects of
kidney function. PAF is likely to act on tubular structures, because
PAF receptor mRNA is present on all segments of the nephron, with
particular abundance in the proximal tubule, comparable to the highest
levels seen in the glomerulus (1). Although it is
presently unclear whether tubule epithelial cells can synthesize PAF,
it is well established that glomerular and renal medullary interstitial
cells can generate PAF (36). Therefore, it is not an
unreasonable expectation that the intrarenal generation of PAF may not
only act locally on glomerular and renal medullary interstitial cells but also gain access to PAF receptors present on the tubular epithelium to influence cellular processes. In addition, circulating PAF may gain
access to proximal tubular cells through glomerular filtration and/or
be potentially synthesized and released from inflammatory blood cell
types infiltrating into the kidney in pathological conditions.
Studies in anesthetized dogs and rats have generally shown that PAF
administration is associated with a decrease in urinary water and
electrolyte excretion that is secondary to PAF-induced falls in
systemic blood pressure, extracellular fluid volume and cardiac output
and/or PAF-induced falls in renal blood flow (RBF) and glomerular
filtration rate (22, 35, 36, 40, 42). Furthermore,
intravenous infusion of PAF at doses that had no effect on systemic and
renal hemodynamics was associated with minimal changes in urinary
excretion variables in anesthetized dogs (40). Similarly,
studies employing isolated rat kidney perfused at constant pressure
have shown that PAF infusion at doses that did not influence renal
vascular resistance or glomerular filtration had no effect on urine
flow rates (UVs) (32). Therefore, there are a number of
reports from whole animal and isolated kidney studies suggesting that
PAF has no measurable effect on tubular transport processes independent
of changes in renal blood pressures and flows. Nevertheless, a direct
action of PAF on renal tubules to modulate transcellular electrolyte
transport has been suggested from measurements of transport function in
microperfused isolated mouse thick ascending limbs, isolated rabbit
cortical collecting tubules, and rat inner medullary collecting duct
cell monolayers (2, 5, 19, 29).
Our initial purpose was to investigate whether PAF has a possible role
to play in the regulation of rat kidney hemodynamic and urinary
excretory function in vivo. The experimental strategy employed in these
experiments was to infuse hypotensive doses of PAF into the renal
artery of anesthetized rats and prevent blood pressure changes being
transmitted to the kidney. This would allow us to 1) address
the effect of PAF on rat renal vascular function, for which there is
considerable confusion in the literature; and 2)
determine whether PAF could influence urinary water and electrolyte excretion independent of blood pressure changes. The contribution of nitric oxide to the effect of PAF in the kidney was
also examined, because nitric oxide can be a secondary mediator of PAF
actions in the cardiovascular system (6, 7, 13, 15, 17).
Our in vivo findings then led us to examine the direct actions of PAF
on freshly isolated rat proximal tubules.
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METHODS |
In vivo studies.
Adult male Wistar rats were anesthetized by an intraperitoneal
injection of pentobarbital sodium (60 mg/kg), and the trachea was
cannulated to allow for a patent air passage. Catheters were then
inserted into 1) the left jugular vein for the intravenous infusion of saline, pressor agents, or periodic injections of diluted
anesthetic at a rate of 45 µl/min; 2) the right carotid artery for systemic blood pressure measurement; 3) the
iliolumbar artery and maneuvered into the aorta and then into the left
renal artery for the intrarenal administration of PAF and other agents at a rate of 55 µl/min; 4) the left femoral artery and
advanced into the aorta just below the iliolumbar artery junction to
estimate renal arterial blood pressure; and 5) the left
ureter for the collection of urine. A silk thread was passed around the
aorta rostoral to the left kidney and attached to a screw device to allow constriction of the aorta and, thereby, regulation of renal arterial blood pressure. The left kidney was surgically and chemically (10% phenol in ethanol) denervated. RBF was recorded with a
noncannulating flow probe placed around the left renal artery and
connected to an electromagnetic flowmeter. A Valco HPLC injection valve
was interspersed into the intrarenal line, allowing the bolus
administration (6 µl) of agents directly into the renal arterial
circulation. Body temperature was monitored and maintained at
~37°C. At the end of all surgical procedures, 5 ml/kg of saline was
administered intravenously over a 2-min period to replace surgical
fluid losses. A minimum of 1 h was allowed for stabilization of
cardiovascular and renal function parameters before the experimental
protocol was begun.
Intrarenal PAF infusion in rats with raised systemic blood
pressure and regulated renal arterial blood pressure.
The experimental protocol consisted of four 25-min periods. A baseline
period was followed by a second period (control) in which AVP was
infused intravenously at 15 ng · min
1 · kg
1
to raise systemic blood pressure and continued at this rate throughout the entire experiment while renal arterial blood pressure was maintained at pre-AVP-infusion levels. PAF was then infused
intrarenally at 2.5 ng · min
1 · kg
1
during the third period (experimental) and then terminated during the
fourth period (recovery). This infusion dose rate of PAF was chosen
because it produces a small fall in systemic blood pressure without
altering RBF (11, 13) and thus provides an online confirmation of the biological activity of PAF. About 10 min before the
start of the second period, the rats were given a combined intravenous
AVP infusion (15 ng · min
1 · kg
1)
and intrarenal N
-nitro-L-arginine
methyl ester (L-NAME) infusion (~0.4 mg/kg bolus + 0.5 ng · min
1 · kg
1),
and this was maintained throughout the entire experiment. Urine was not collected during the first 5 min of a 25-min period so that
preformed urine would escape the dead space in the collection system.
At the end of each experiment, an in vivo calibration of the flow probe
was undertaken using the left renal artery or left femoral artery and
collecting timed blood samples.
In vitro studies.
Proximal tubules were isolated from the renal cortex of anesthetized
male Wistar rats by in vivo and in vitro collagenase digestion and
Percoll density centrifugation. Dissociated tubules were placed in a
closed, thermoregulated chamber, and tissue oxygen consumption
(QO2) was measured by a Clarke oxygen
electrode. All procedures have been previously described in detail
(12). QO2 can be used as an online
integrated index of sodium transport activity because of the tight
coupling between Na+-K+-ATPase activity and
mitochondrial oxidative phosphorylation (23).
In experiments using receptor antagonists or signaling pathway
inhibitors, these were added to the proximal tubule suspension (preincubated for 10-30 min) and chamber. All other drugs were added as 25-µl boluses to the tubule-containing chamber via its injection port. To minimize the variability of
QO2 from different tubule preparations (basal
and nystatin-stimulated QO2 averaged 34 and 57 nmol
O2 · min
1 · mg
protein
1, respectively), the effects of drug treatments
were expressed as a percent change from baseline values. All drug
solutions were prepared fresh daily, and their molar concentrations
indicate the final concentrations achieved in the chamber.
The following experiments were performed in rat proximal tubule
suspensions. First, concentration-QO2 response
curves were generated for PAF in untreated and nystatin-treated
proximal tubules to ascertain the sensitivity and membrane location of
PAF actions. Second, whether the actions of PAF were mediated by a PAF
receptor and/or related to a PAF metabolite was determined. Third,
whether the actions of PAF were due to an effect on cell respiratory
processes and/or active Na+-K+-ATPase activity
was examined. Fourth, possible intracellular signaling pathways
mediating the functional effects of PAF on proximal tubule
QO2 were identified.
Drugs.
We received a gift of BN-52021 (Institut Henri Beaufour).
L-
-phosphatidylcholine,
-acetyl-
-O-hexadecyl (PAF),
D-
-phosphatidylcholine,
-acetyl-
-O-hexadecyl (D-PAF),
L-
-lysophosphatidylcholine,
-acetyl-
-O-hexadecyl (lyso-PAF),
L-NAME, 17-octadecynoic acid, and all other drugs were
purchased from Sigma.
Statistics.
Data are shown as means ± SE. Multiple groups were analyzed by
one-way ANOVA with an appropriate post hoc test. Significance within a
group was analyzed by a paired Student's t-test and between two groups using an unpaired Student's t-test. Statistical
significance was taken as a P value
0.05.
 |
RESULTS |
In vivo studies.
Results obtained from AVP- and PAF-infused rats in the absence or
presence of L-NAME (nitric oxide synthase inhibitor) are shown in Tables 1 and
2, respectively. As shown in Table 1, intravenous AVP infusions elevated mean arterial blood pressure (MAP)
during which renal arterial blood pressure (RPP) was effectively regulated at pre-AVP-infusion levels and RBF, UV, urinary sodium excretion (UNaV), and urinary potassium excretion
(UKV) remained unchanged. Intrarenal PAF infusion at 2.5 ng · min
1 · kg
1
resulted in a small fall in MAP, no change in RPP, and increased RBF
(Fig. 1), UV, UNaV, and
UKV by 17.0 ± 1.7, 62.1 ± 14.4, 188.2 ± 38.9, and 46.8 ± 13.4%, respectively. Complete recovery
of MAP, RBF, and UKV were observed on cessation of the PAF
infusion, whereas UV and UNaV remained elevated. Data in
Table 2 show that MAP increased during simultaneous AVP and
L-NAME infusion, which tended to be greater than that
observed in rats infused with AVP alone. RPP was effectively regulated
at pre-AVP/L-NAME infusion levels and RBF, UV,
UNaV, and UKV fell by 29.7 ± 1.8, 41.7 ± 6.6, 32.3 ± 17.7, and 48.8 ± 6.5%,
respectively. Intrarenal infusion of PAF at 2.5 ng · min
1 · kg
1
did not alter MAP or RPP and was associated with a very small decrease
in RBF (~5%) that continued to fall after termination of the PAF
infusion and likely reflected a failure to achieve a plateau phase
during nitric oxide synthase inhibition. The PAF infusion period was
also associated with an increase in UNaV and UKV of 113.5 ± 41.1 and 36.7 ± 13.2%,
respectively, as well as a trend toward an increased UV
(P = 0.054, one-way ANOVA and Student's t-test). Urinary water and solute excretion rates remained
elevated on cessation of the PAF infusion.

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Fig. 1.
Effect of intrarenal platelet-activating factor (PAF)
infusion on systemic blood pressure [mean arterial blood pressure
(MAP)], renal arterial blood pressure (RAP), and total renal blood
flow (RBF). Rats were given an intravenous infusion of AVP to raise
mean arterial blood pressure while constricting the aorta above both
kidneys to maintain renal arterial blood pressure constant.
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Figure 2 depicts data showing that the
PAF-induced rise in urinary water excretion rate and UNaV
was significantly reduced in L-NAME-treated rats, with a
similar trend observed for UKV (P = 0.082 using an unpaired Student's t-test). Similar RBF and urinary excretory responses were observed in the absence or presence of
L-NAME during an intrarenal infusion of PAF at the higher
dose rate of 10 ng · min
1 · kg
1
(not shown).

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Fig. 2.
Change in urinary flow (UV), urinary sodium excretion
(UNaV), and urinary potassium excretion rates
(UKV) during intrarenal PAF infusion in the absence or
presence of the nitric oxide synthesis inhibitor
N -nitro-L-arginine methyl ester
(L-NAME). *P < 0.05 from the corresponding
response in the absence of L-NAME using an unpaired
Student's t-test.
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In vitro studies.
Addition of PAF to nontreated fresh suspensions of rat proximal tubules
resulted in a concentration-dependent inhibition of basal
QO2 with a threshold concentration between 0.1 and 1 nM (Fig. 3). To examine whether PAF
interfered with sodium exit from the proximal tubule cell, we treated
cells with nystatin (sodium ionophore), which allows sodium to freely
enter the cell and results in ~70% increase in
QO2 due to enhanced basolateral
Na+-K+-ATPase activity. Under
nystatin-stimulated conditions, the proximal tubules were more
sensitive to the inhibitory actions of PAF, with a threshold
concentration of between 1 and 10 pM (Fig. 3). These results suggested
that one action of PAF could be to inhibit basolateral
Na+-K+-ATPase activity. Subsequent studies
employed 1 nM PAF because this concentration produced a near maximal
inhibition of proximal tubule QO2.

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Fig. 3.
Percent reduction in proximal tubule O2
consumption (PT QO2) induced by PAF in control
or nystatin-stimulated proximal tubules. Values are means of 4-11
separate measurements.
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Figure 4 shows that only the levorotatory
isomer of PAF was capable of inhibiting nystatin-stimulated proximal
tubule QO2 whereas vehicle and the
dextrorotatory isomer of PAF (D-PAF) had no effect. This
suggested that PAF acts through a stereospecific receptor site to exert
its inhibitory effect on QO2. We also found that the major product of PAF metabolism, lyso-PAF, did not alter nystatin-stimulated QO2, implying that PAF
itself was responsible for the observed biologial response (Fig. 4).

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Fig. 4.
Effect of the levorotatory (L-PAF, n = 10) and dextrorotatory (D-PAF, n = 9) stereoisomers of
PAF, lyso-PAF (n = 8), and vehicle (methanol/chloroform
diluted appropriately in BSA-containing saline, n = 4).
All PAF concentrations were 1 nM. ***P < 0.001 from
the proximal tubule QO2 value before the
addition of PAF using a paired Student's t-test. Also
depicted is the ability of 1 µM BN-52021 (PAF receptor antagonist,
n = 10) to antagonize the 1 nM PAF-induced reduction in
proximal tubule QO2. #P < 0.001 from the corresponding PAF response (n = 10) in
the absence of PAF receptor blockade using an unpaired Student's
t-test. Values are number of separate measurements.
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Addition of 5 µM FCCP (mitochondrial oxidative phosphorylation
uncoupler) to control proximal tubules increased basal
QO2 by 217 ± 42% (P < 0.05, n = 4). In a separate group of experiments, FCCP
caused a similar increase in basal QO2 of
198 ± 22% (P < 0.001, n = 8),
and the subsequent addition of 1 nM PAF was without effect (
2 ± 2%, n = 8). Conversely, control proximal tubules treated with 1 nM PAF decreased basal QO2 by
27 ± 7% (P < 0.05, n = 5) and
did not impair the increase in QO2 on
subsequent addition of FCCP (237 ± 27%, P < 0.001, n = 5). These findings suggest that the
inhibitory effect of PAF on proximal tubule function was not due to an
inhibition of mitochondrial respiratory activity. Treating proximal
tubules with 5 mM ouabain (Na+-K+-ATPase
inhibitor) reduced basal QO2 by 33 ± 5%
(P < 0.001, n = 10) and abolished the
stimulatory action of nystatin, confirming that the nystatin effect on
QO2 was by increasing
Na+-K+-ATPase activity. Conversely, ouabain
reduced nystatin-stimulated proximal tubule QO2
by 58 ± 2% (P < 0.001, n = 9)
and abolished the inhibitory action of PAF. Together, these findings
indicate that PAF had direct actions on proximal tubule epithelium and that at least one effect was to suppress an ouabain-inhibitable component of transcellular sodium transport, namely, basolateral Na+-K+-ATPase pump activity.
To pharmacologically identify whether a receptor mediated the tubular
action of PAF, we examined the ability of the phospholipid to inhibit
nystatin-stimulated cellular transport in tubules preincubated with the
PAF receptor antagonist BN-52021. As depicted in Fig. 4, we found that
the inhibitory action of PAF on proximal tubule QO2 was abolished by 1 µM BN-52021. This
effect of BN-52021 appeared to be specifically related to blocking the
PAF receptor, because it did not effect the inhibitory action of 1 pM
ANG IV on nystatin-stimulated proximal tubule
QO2 (ANG IV, 19.5 ± 1.9%, vs. ANG
IV + BN-52021, 18.2 ± 0.6%, n = 2 each), an
ANG IV response known to be mediated by the angiotensin AT4
receptor (14).
We then examined possible intracellular signaling mechanisms involved
in the inhibitory action of PAF on tubule QO2.
The contribution of the nitric oxide-guanylate cyclase pathway was
assessed by preincubating proximal tubules with either 10 µM
methylene blue (guanylate cyclase inhibitor) or 100 µM
L-NAME (nitric oxide synthase inhibitor). Curiously, we
observed that the increase in QO2 on nystatin
administration was quickly dissipated in tubules pretreated with
methylene blue only. However, both methylene blue and
L-NAME treatments did not interfere with the inhibitory
action of PAF on nystatin-stimulated proximal tubule
QO2 (Fig. 5). In
contrast, phospholipase A2 (PLA2) inhibition
with quinacrine (data for 0.1 and 1 mM were similar and combined),
cytochrome-P-450 monooxygenase inhibition with SKF-525A (50 µM proadifen) or 17-octadecynoic acid (specific suicide inhibitor of
cytochrome-P-450 fatty acid
-hydroxylase, data for 1 and
10 µM were similar and combined) resulted in a 40-60% reduction
in the effect of PAF on nystatin-stimulated proximal tubule
QO2 (Fig. 5). Basal and
nystatin-stimulated QO2 were similar in all
drug-incubated tissues (the only exception was a depressed nystatin
response in methylene blue-treated tissues) compared with untreated
control tissue.

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Fig. 5.
Lack of effect of 10 µM metheylene blue (MB; guanylate
cyclase inhibitor, n = 8) or 100 µM
L-NAME (nitric oxide synthase inhibitor, n = 10) to alter the reduction in proximal tubule
QO2 induced by 1 nM PAF (n = 12). Reduction in proximal tubule QO2 by 1 nM
PAF (n = 11) and its modification by 0.1-1 mM
quinacrine (phospholipase A2 inhibitor, n = 9), 50 µM SKF-525A (cytochrome-P-450 monooxygenase
inhibitor, n = 9), and 1-10 µM 17-octadecynoic
acid (17-ODYA; cytochrome-P-450 fatty acid -hydroxylase
inhibitor, n = 14). *P < 0.05 from PAF
control using one-way ANOVA and Dunnett's test. Values are number of
separate measurements.
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 |
DISCUSSION |
Early reports from our laboratory demonstrated that PAF
administered as a bolus (10) or infusion (11)
into the renal artery of anesthetized rats resulted in a decrease in
renal vascular resistance and reactivity, and we had speculated on the
involvement of endothelium-derived relaxing factor (nitric oxide) in
these PAF-mediated processes. Later, we demonstrated an important role for nitric oxide in the PAF-induced attenuation of ANG II-mediated vasoconstriction in the rat renal vascular bed (13). The
present study also suggests that nitric oxide contributes to the renal vasodilatory and systemic hypotensive responses to intrarenal PAF
infusion in the rat.
An intravenous infusion of AVP was used to raise systemic blood
pressure and had minimal impact on RBF, highlighting the known insensitivity of the rat renal vasculature to the vasoconstrictor influences of intravenous AVP infusions (33, 41).
Recently, it was proposed that renal sympathoinhibition accounts for
the inability of intravenous pressor doses of AVP to decrease RBF in
conscious rats, because renal denervation unmasked a vasopressin V1 receptor-mediated fall in RBF (33). Our
findings in anesthetized rats would not support this proposal, because
we found no evidence of an AVP-mediated decrease in RBF under
conditions in which the associated increase in blood pressure was
prevented from being transmitted to the denervated kidney. Therefore,
we conclude that it is unlikely that the renal vasodilation induced by
PAF was simply due to antagonism of a renal vasoconstrictor effect of AVP. In addition, PAF is a relatively poor inhibitor of the renal vasoconstriction induced by the intrarenal injection of high
pharmacological doses of AVP (11). In previous studies, we
could only show weak and transient renal vasodilatory responses to
nonhypotensive intrarenal infusion rates of PAF (11).
However, the experimental setup employed in the present study
(preventing the PAF-induced systemic hypotension and associated
increase in sympathetic outflow from being transmitted to the
denervated kidney) allowed us to reveal a strong and sustained kidney
vasodilatory response to intrarenal PAF infusions in normal rats in vivo.
To our knowledge, the present results are the first to demonstrate that
PAF infusion into the rat renal artery can be associated with a
significant diuresis, natriuresis, and kaliuresis, the magnitude of
which was attenuated by nitric oxide synthase inhibition. However,
opposing our in vivo findings is the report that the intrarenal
infusion of PAF into anesthetized rats resulted in a decrease in
urinary water excretion rate and UNaV that was largely due
to PAF-induced falls in RBF and glomerular filtration rate (42). Although we cannot readily explain these hemodynamic
discrepancies, it should be noted that a number of investigators have
demonstrated that PAF can possess vasodilatory properties in the kidney
(10, 17; see also citations in Ref. 11) and
inhibit solute reabsorptive processes by acting at the level of the
nephron (2, 5, 19, 29). In the present study, we regulated
renal arterial blood pressure such that the AVP-induced rise in
systemic blood pressure was not transmitted to the denervated kidney
and found that urinary water and solute excretion remained unchanged.
The actions of AVP in the kidney have been reported to range from a
decrease to an increase in urinary water excretion rate and
UNaV, as well as any conceivable combination of urinary
excretory responses between these two extremes (3, 8, 9, 18,
20). This highlights the complex and multifactorial nature of
AVP actions in the kidney to regulate urinary water and solute
excretion, including integrated effects on blood pressure,
sympathoinhibition, renal hemodynamics, and tubular function (3,
8, 9, 18, 20). Although one would expect the direct stimulatory
actions of AVP on tubular transport processes to result in a decrease in urinary water excretion rate and UNaV (3),
investigators have also reported that nonpressor doses of AVP infused
into the kidney can be associated with no change in both renal
hemodynamics and urinary water excretion rate and/or UNaV
(8, 20). Consequently, we conclude that the subsequent
intrarenal infusion of PAF was likely responsible for the observed
increase in urinary water and solute excretion in the anesthetized rat.
In addition to a significant role for nitric oxide in the glomerular
and vascular actions of PAF in the kidney (13, 17, 24),
the renal nitric oxide pathway is also known to be natriuretic by
influencing several kidney systems, including the cortical and
medullary microcirculation, interstitial hydrostatic pressure, and
tubular transport processes (21, 25, 37). In the present
study, blockade of nitric oxide synthesis was found to dramatically
attenuate the ability of PAF to elicit a diuresis, natriuresis, and
kaliuresis and suggests either a direct or indirect role of nitric
oxide in these urinary water and electrolyte responses.
Because our in vivo results do not allow us to dissociate the urinary
excretory responses from the PAF-mediated renal hemodynamic effects, we
examined the direct actions of PAF on isolated rat proximal tubules,
because this nephron segment is a major site for the reabsorption of
sodium and water and contains abundant PAF receptor mRNA
(1). No information is available on the direct actions of
PAF on this segment of the nephron. We found that PAF inhibited
proximal tubule QO2 without influencing
mitochondrial uncoupled QO2 rates, suggesting a
reduction in energy-dependent transcellular solute transport. Proximal
tubules were treated with nystatin (sodium ionophore that functionally
bypasses the rate-limiting step of sodium entry into the cell),
allowing an indirect assessment of sodium efflux from the cell via the
basolateral Na+-K+-ATPase pump
(23). Nystatin treatment increased the sensitivity of the
proximal tubules to the inhibitory actions of PAF, suggesting that the
lipid may act to reduce basolateral
Na+-K+-ATPase pump activity. This conclusion is
supported by the observation that ouabain, an
Na+-K+-ATPase inhibitor, prevented the
inhibitory effect of PAF on nystatin-stimulated proximal tubule
QO2, as well as several reports that PAF can
inhibit membrane Na+-K+-ATPase activity in a
number of cell types (4, 16). In addition, endogenous PAF
was found to contribute to the decrease in renal cortex
Na+-K+-ATPase activity after reperfusion of the
ischemic rat small intestine, an effect that could be blocked
by the PAF receptor antagonist BN-52021 (39). Our results
obtained with D-PAF, lyso-PAF, and PAF in the presence of
BN-52021 would also be consistent with a PAF receptor mediating the
biological response of PAF in isolated rat proximal tubules.
The dilatory action of PAF on the renal vasculature is largely
dependent on the nitric oxide pathway (present study and Ref. 17), and others have shown that activation of guanylate
cyclase and the subsequent rise in cGMP mediate many actions of nitric oxide in the body (26). We found that nitric oxide
blockade with L-NAME attenuated the natriuretic and
diuretic response to intrarenal PAF infusion in the rat in vivo. This
reduced urinary excretory response could be due to L-NAME
preventing PAF-induced renal vasodilation and/or reducing a
natriuretic/diuretic action of PAF at the level of the nephron. Both
nitric oxide and cGMP can be produced in the proximal tubule, with both
capable of inhibiting proximal tubule
Na+-K+-ATPase activity (21).
However, we found that methylene blue and L-NAME at
concentrations known to inhibit tubular guanylate cyclase and nitric
oxide synthase activity, respectively, did not interfere with the
inhibitory actions of PAF on rat proximal transport processes. This
would seem to rule out nitric oxide as a candidate for mediating the
direct inhibitory effect of PAF on proximal tubule solute reabsorptive
function. Although it is presently unknown whether PAF can stimulate
nitric oxide biosynthesis and/or guanylate cyclase activity in the
proximal tubule, the phospholipid can stimulate cGMP in glomerular
cells when coincubated with endothelial cells (24) and
inhibit solute reabsorptive function of the medullary thick ascending
limb (mTAL) via a cGMP-dependent pathway (29). Our results
do not rule out the possibility that PAF-stimulated nitric oxide/cGMP
synthesis from the renal microcirculation and other nonproximal tubule
sources may inhibit proximal tubule solute reabsorptive processes in
vivo (21). Further downstream of the proximal tubule,
investigators have demonstrated that PAF can reduce the ability of
vasopressin to increase transepithelial resistance (a measure of active
solute reabsorption) in cultured rat inner medullary collecting duct
cells (19) or microperfused rabbit cortical collecting
ducts (5). This would likely result in a natriuresis and
diuresis in vivo, because this terminal segment of the nephron is
important in regulating the final amount of sodium and water that is
excreted in the urine.
The arachidonic acid pathway appeared to be of major importance in the
direct actions of PAF in the rat proximal tubule. Inhibitors of
PLA2 and cytochrome-P-450 monooxygenase were
able to significantly attenuate the inhibitory actions of PAF on
proximal tubule QO2. This is similar to the
proposed pathway by which a number of renal hormones inhibit proximal
tubule Na+-K+-ATPase activity
(34). PAF is a potent activator of PLA2,
resulting in the formation of arachidonic acid (15, 28,
36). Processing of arachidonic acid by
cytochrome-P-450 monooxygenase can lead to the formation of
HETEs and epoxyeicosatrienoic acids that can reduce the transcellular
movement of sodium across the proximal tubule; e.g., 20-HETE inhibits
basolateral Na+-K+-ATPase activity through a
PKC-dependent phosphorylation of the
-subunit of ATPase, whereas
epoxyeicosatrienoic acids are natriuretic perhaps by inhibiting the
translocation of the Na+-H+ antiporter to the
apical membrane (34). Although PLA2 and
cytochrome-P-450 monooxygenase products do not appear to be
responsible for the inhibitory effect of PAF on solute transport
processes in the mouse mTAL (2), others have implicated
the cytochrome-P-450 monooxygenase pathway in the cellular
biological actions of PAF in the rat hindlimb (38) and
human neutrophil (27). Our results would suggest that one
direct action of PAF on the rat proximal tubule is to decrease sodium
transport reabsorption via a PLA2 and
cytochrome-P-450 monooxygenase pathway and may reflect the fact that intracellular signaling systems activated by PAF in the
nephron are site specific. However, our results did not reveal a major
contribution of nonnitric oxide renal tubule pathways in the renal
excretory responses to intrarenal PAF infusion in nitric oxide synthase
inhibitor-treated rats in vivo. This is probably related to the fact
that infusing PAF into the renal vascular compartment greatly limits
its access to renal tubules (30). PAF is not only a
circulating lipid but is also synthesized by glomerular cells, renal
medullary interstitial cells, and inflammatory cells infiltrating the
kidney that would allow both greater access and higher concentrations
of PAF at renal tubule sites. Presumably, this intrarenal
generation/release of PAF would allow tubule PAF receptor systems to
have a greater role in regulating renal excretory function. On the
other hand, we cannot exclude the possibility that the multiple and
complex changes in renal function induced by nitric oxide synthase
inhibition may mask/attenuate the contribution of the renal tubules to
the PAF-induced diuresis, natriuresis, and kaliuresis.
In conclusion, a number of factors may potentially contribute to the
PAF-induced increase in urinary water and electrolyte excretion
observed in vivo and include 1) PAF-induced increase in
kidney blood flow that was secondarily mediated by nitric oxide, 2) perhaps PAF-stimulated nitric oxide synthesis from a
nonproximal tubule source leading to a reduction in proximal tubule
sodium reabsorption, 3) PAF activation of proximal tubule
PLA2 and cytochrome-P-450 monooxygenase pathways
to attenuate sodium reabsorption processes, and 4)
PAF-mediated inhibition of solute reabsorption in the mTAL and
collecting ducts.
 |
ACKNOWLEDGEMENTS |
This work was partly supported by an RJR-Leon Golberg Research
Award in Pharmacology and Toxicology.
 |
FOOTNOTES |
Reprint requests and other correspondence: R. K. Handa (E-mail: rajash_handa{at}hotmail.com).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00117.2002
Received 25 March 2002; accepted in final form 11 September 2002.
 |
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