Platelet-activating factor and solute transport processes inthe kidney - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:32 |只看该作者 |倒序浏览 |打印

作者：Rajash K.Handa, Jack W.Strandhoy, CarlosE.Giammattei,  Shelly E.Handa作者单位：Department of Physiology and Pharmacology, Wake ForestUniversity School of Medicine, Winston-Salem, North Carolina 27157 - ~; g8 k6 {/ k  G. k3 ?6 }

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      【摘要】: x4 d7 t  M+ |! ~3 \
   We examined thehemodynamic and tubular transport mechanisms by whichplatelet-activating factor (PAF) regulates salt and water excretion. Inanesthetized, renally denervated male Wistar rats, with raised systemicblood pressure and renal arterial blood pressure maintained at normallevels, intrarenal PAF infusion at 2.5 ng · min 1 · kg 1 resulted in a small fall in systemic blood pressure (no change in renalarterial blood pressure) and an increase in renal blood flow andurinary water, sodium, and potassium excretion rates. The PAF-inducedchanges in cardiovascular and renal hemodynamic function were abolishedand renal excretory function greatly attenuated by treating rats with anitric oxide synthase inhibitor. To determine whether a tubular site ofaction was involved in the natriuretic effect of PAF, cortical proximaltubules were enzymatically dissociated from male Wistar rat kidneys,and oxygen consumption rates (Q O 2 ) were used asan integrated index of transcellular sodium transport. PAF at 1 nMmaximally inhibited Q O 2 in both untreated andnystatin-stimulated (sodium entry into renal cell is not rate limiting)proximal tubules by ~20%. Blockade of PAF receptors orNa -K -ATPase pump activity with BN-52021 orouabain, respectively, abolished the effect of PAF onnystatin-stimulated proximal tubule Q O 2.Inhibition of nitric oxide synthase or guanylate cyclase systems didnot alter PAF-mediated inhibition of nystatin-stimulated proximaltubule Q O 2, whereas phospholipaseA 2 or cytochrome- P -450 monooxygenase inhibitionresulted in a 40-60% reduction. These findings suggest thatstimulation of PAF receptors on the proximal tubule decreasestranscellular sodium transport by activating phospholipaseA 2 and the cytochrome- P -450 monooxygenasepathways that lead to the inhibition of an ouabain-sensitive component of the basolateral Na -K -ATPase pump. Thus PAFcan activate both an arachidonate pathway-mediated suppression ofproximal tubule sodium transport and a nitric oxide pathway-mediateddilatory action on renal hemodynamics that likely contributes to thenatriuresis and diuresis observed in vivo. , _! @( U5 @, S, w, \  Y8 R
      【关键词】 nitric oxide vasopressin blood pressure renal blood flow urinary water electrolyte excretion guanylate cyclase phospholipaseA cytochrome P monooxygenase sodiumpotassiumadenosine ‘triphosphatase proximal tubule oxygenconsumption rat
   INTRODUCTION

PLATELET-ACTIVATING FACTOR (PAF)represents a group of1-alkyl-2-acetyl- sn -glycero-3-phosphocholines that is aclass of lipid mediators involved in many physiological (e.g.,cell-cell signaling, regulation of blood pressure, and reproductionbiology) and pathological (e.g., endotoxic/septic shock, immunologicaland inflammatory diseases, and ischemia and reperfusion injury)processes in the body ( 15, 28 ). Much of the research onPAF and the kidney has focused on the role of PAF in renal vascular andglomerular function. This is understandable given that PAF receptorantagonists can improve the outcome of many kidney diseases with avascular or glomerular etiology ( 22, 31 ). Consequently,little is known about the possible actions of PAF on other aspects ofkidney function. PAF is likely to act on tubular structures, becausePAF receptor mRNA is present on all segments of the nephron, withparticular abundance in the proximal tubule, comparable to the highestlevels seen in the glomerulus ( 1 ). Although it ispresently unclear whether tubule epithelial cells can synthesize PAF,it is well established that glomerular and renal medullary interstitialcells can generate PAF ( 36 ). Therefore, it is not anunreasonable expectation that the intrarenal generation of PAF may notonly 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 gainaccess to proximal tubular cells through glomerular filtration and/orbe potentially synthesized and released from inflammatory blood celltypes infiltrating into the kidney in pathological conditions.; C. f; U* ]( Z/ T/ J# T3 @1 G6 K

Studies in anesthetized dogs and rats have generally shown that PAFadministration is associated with a decrease in urinary water andelectrolyte excretion that is secondary to PAF-induced falls insystemic blood pressure, extracellular fluid volume and cardiac outputand/or PAF-induced falls in renal blood flow (RBF) and glomerularfiltration rate ( 22, 35, 36, 40, 42 ). Furthermore,intravenous infusion of PAF at doses that had no effect on systemic andrenal hemodynamics was associated with minimal changes in urinaryexcretion variables in anesthetized dogs ( 40 ). Similarly,studies employing isolated rat kidney perfused at constant pressurehave shown that PAF infusion at doses that did not influence renalvascular resistance or glomerular filtration had no effect on urineflow rates (UVs) ( 32 ). Therefore, there are a number ofreports from whole animal and isolated kidney studies suggesting thatPAF has no measurable effect on tubular transport processes independentof changes in renal blood pressures and flows. Nevertheless, a directaction of PAF on renal tubules to modulate transcellular electrolytetransport has been suggested from measurements of transport function inmicroperfused isolated mouse thick ascending limbs, isolated rabbitcortical collecting tubules, and rat inner medullary collecting ductcell monolayers ( 2, 5, 19, 29 ).
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Our initial purpose was to investigate whether PAF has a possible roleto play in the regulation of rat kidney hemodynamic and urinaryexcretory function in vivo. The experimental strategy employed in theseexperiments was to infuse hypotensive doses of PAF into the renalartery of anesthetized rats and prevent blood pressure changes beingtransmitted to the kidney. This would allow us to 1 ) addressthe effect of PAF on rat renal vascular function, for which there isconsiderable 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 wasalso examined, because nitric oxide can be a secondary mediator of PAFactions in the cardiovascular system ( 6, 7, 13, 15, 17 ).Our in vivo findings then led us to examine the direct actions of PAFon freshly isolated rat proximal tubules.
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In vivo studies. Adult male Wistar rats were anesthetized by an intraperitonealinjection of pentobarbital sodium (60 mg/kg), and the trachea wascannulated to allow for a patent air passage. Catheters were theninserted into 1 ) the left jugular vein for the intravenous infusion of saline, pressor agents, or periodic injections of dilutedanesthetic at a rate of 45 µl/min; 2 ) the right carotid artery for systemic blood pressure measurement; 3 ) theiliolumbar artery and maneuvered into the aorta and then into the leftrenal artery for the intrarenal administration of PAF and other agents at a rate of 55 µl/min; 4 ) the left femoral artery andadvanced into the aorta just below the iliolumbar artery junction toestimate renal arterial blood pressure; and 5 ) the leftureter for the collection of urine. A silk thread was passed around theaorta 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 anoncannulating flow probe placed around the left renal artery andconnected to an electromagnetic flowmeter. A Valco HPLC injection valvewas interspersed into the intrarenal line, allowing the bolusadministration (6 µl) of agents directly into the renal arterialcirculation. Body temperature was monitored and maintained at~37°C. At the end of all surgical procedures, 5 ml/kg of saline wasadministered intravenously over a 2-min period to replace surgicalfluid losses. A minimum of 1 h was allowed for stabilization ofcardiovascular and renal function parameters before the experimentalprotocol was begun./ M1 b6 f5 I) R" ^4 |7 ?

Intrarenal PAF infusion in rats with raised systemic bloodpressure and regulated renal arterial blood pressure. The experimental protocol consisted of four 25-min periods. A baselineperiod was followed by a second period (control) in which AVP wasinfused 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 infusedintrarenally at 2.5 ng · min 1 · kg 1 during the third period (experimental) and then terminated during thefourth period (recovery). This infusion dose rate of PAF was chosenbecause it produces a small fall in systemic blood pressure withoutaltering RBF ( 11, 13 ) and thus provides an online confirmation of the biological activity of PAF. About 10 min before thestart of the second period, the rats were given a combined intravenousAVP infusion (15 ng · min 1 · kg 1 )and intrarenal N -nitro- L -argininemethyl 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 thatpreformed urine would escape the dead space in the collection system.At the end of each experiment, an in vivo calibration of the flow probewas undertaken using the left renal artery or left femoral artery andcollecting timed blood samples.
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In vitro studies. Proximal tubules were isolated from the renal cortex of anesthetizedmale Wistar rats by in vivo and in vitro collagenase digestion andPercoll density centrifugation. Dissociated tubules were placed in aclosed, thermoregulated chamber, and tissue oxygen consumption(Q O 2 ) was measured by a Clarke oxygenelectrode. All procedures have been previously described in detail( 12 ). Q O 2 can be used as an onlineintegrated index of sodium transport activity because of the tightcoupling between Na -K -ATPase activity andmitochondrial oxidative phosphorylation ( 23 ).# L" Z% Y3 O! [9 R0 W+ ]( H1 ?

In experiments using receptor antagonists or signaling pathwayinhibitors, 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 ofQ O 2 from different tubule preparations (basaland nystatin-stimulated Q O 2 averaged 34 and 57 nmolO 2 · min 1 · mgprotein 1, respectively), the effects of drug treatmentswere expressed as a percent change from baseline values. All drugsolutions were prepared fresh daily, and their molar concentrationsindicate the final concentrations achieved in the chamber.
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The following experiments were performed in rat proximal tubulesuspensions. First, concentration-Q O 2 responsecurves were generated for PAF in untreated and nystatin-treatedproximal tubules to ascertain the sensitivity and membrane location ofPAF actions. Second, whether the actions of PAF were mediated by a PAFreceptor and/or related to a PAF metabolite was determined. Third,whether the actions of PAF were due to an effect on cell respiratoryprocesses and/or active Na -K -ATPase activitywas examined. Fourth, possible intracellular signaling pathwaysmediating the functional effects of PAF on proximal tubuleQ O 2 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 werepurchased from Sigma.4 }* F! u/ T9 M
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Statistics. Data are shown as means ± SE. Multiple groups were analyzed byone-way ANOVA with an appropriate post hoc test. Significance within agroup was analyzed by a paired Student's t -test and between two groups using an unpaired Student's t -test. Statisticalsignificance was taken as a P value 0.05.# c$ i6 M# g; {+ H: q' U  n
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RESULTS

In vivo studies. Results obtained from AVP- and PAF-infused rats in the absence orpresence 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 (U Na V), and urinary potassium excretion(U K V) 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, U Na V, andU K V by 17.0 ± 1.7, 62.1 ± 14.4, 188.2 ± 38.9, and 46.8 ± 13.4%, respectively. Complete recoveryof MAP, RBF, and U K V were observed on cessation of the PAFinfusion, whereas UV and U Na V remained elevated. Data inTable 2 show that MAP increased during simultaneous AVP and L -NAME infusion, which tended to be greater than thatobserved in rats infused with AVP alone. RPP was effectively regulatedat pre-AVP/ L -NAME infusion levels and RBF, UV,U Na V, and U K V 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 decreasein RBF (~5%) that continued to fall after termination of the PAFinfusion and likely reflected a failure to achieve a plateau phaseduring nitric oxide synthase inhibition. The PAF infusion period wasalso associated with an increase in U Na V and U K V 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 remainedelevated on cessation of the PAF infusion.. ], o6 `4 s, [0 K0 u. z' ^: M

Table 1. Renal effects of PAF (2.5 ng · min 1 · kg 1 )in AVP-treated rats with RPP held constant
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Table 2. Renal effects of PAF (2.5 ng · min 1 · kg 1 )in AVP- and L -NAME-treated rats with RPP held constant
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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 bloodflow (RBF). Rats were given an intravenous infusion of AVP to raisemean arterial blood pressure while constricting the aorta above bothkidneys to maintain renal arterial blood pressure constant.

Figure 2 depicts data showing that thePAF-induced rise in urinary water excretion rate and U Na Vwas significantly reduced in L -NAME-treated rats, with asimilar trend observed for U K V ( 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 higherdose rate of 10 ng · min 1 · kg 1 (not shown).
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Fig. 2. Change in urinary flow (UV), urinary sodium excretion(U Na V), and urinary potassium excretion rates(U K V) during intrarenal PAF infusion in the absence orpresence of the nitric oxide synthesis inhibitor N -nitro- L -arginine methyl ester( L -NAME). * P L -NAME using an unpairedStudent's t -test.
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In vitro studies. Addition of PAF to nontreated fresh suspensions of rat proximal tubulesresulted in a concentration-dependent inhibition of basalQ O 2 with a threshold concentration between 0.1 and 1 nM (Fig. 3 ). To examine whether PAFinterfered with sodium exit from the proximal tubule cell, we treatedcells with nystatin (sodium ionophore), which allows sodium to freelyenter the cell and results in ~70% increase inQ O 2 due to enhanced basolateralNa -K -ATPase activity. Undernystatin-stimulated conditions, the proximal tubules were moresensitive to the inhibitory actions of PAF, with a thresholdconcentration of between 1 and 10 pM (Fig. 3 ). These results suggestedthat one action of PAF could be to inhibit basolateralNa -K -ATPase activity. Subsequent studiesemployed 1 nM PAF because this concentration produced a near maximalinhibition of proximal tubule Q O 2.
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Fig. 3. Percent reduction in proximal tubule O 2 consumption (PT Q O 2 ) induced by PAF in controlor nystatin-stimulated proximal tubules. Values are means of 4-11separate measurements.

Figure 4 shows that only the levorotatoryisomer of PAF was capable of inhibiting nystatin-stimulated proximaltubule Q O 2 whereas vehicle and thedextrorotatory isomer of PAF ( D -PAF) had no effect. Thissuggested that PAF acts through a stereospecific receptor site to exertits inhibitory effect on Q O 2. We also found that the major product of PAF metabolism, lyso-PAF, did not alter nystatin-stimulated Q O 2, implying that PAFitself was responsible for the observed biologial response (Fig. 4 ).! O3 F& ^6 }, d3 _7 H
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Fig. 4. Effect of the levorotatory (L-PAF, n = 10) and dextrorotatory (D-PAF, n = 9) stereoisomers ofPAF, lyso-PAF ( n = 8), and vehicle (methanol/chloroformdiluted appropriately in BSA-containing saline, n = 4).All PAF concentrations were 1 nM. *** P O 2 value before theaddition of PAF using a paired Student's t -test. Alsodepicted is the ability of 1 µM BN-52021 (PAF receptor antagonist, n = 10) to antagonize the 1 nM PAF-induced reduction inproximal tubule Q O 2. # P n = 10) inthe absence of PAF receptor blockade using an unpaired Student's t -test. Values are number of separate measurements.3 P* L: J; S: n4 V! e6 d3 [

Addition of 5 µM FCCP (mitochondrial oxidative phosphorylationuncoupler) to control proximal tubules increased basalQ O 2 by 217 ± 42% ( P n = 4). In a separate group of experiments, FCCPcaused a similar increase in basal Q O 2 of198 ± 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 Q O 2 by27 ± 7% ( P n = 5) anddid not impair the increase in Q O 2 onsubsequent addition of FCCP (237 ± 27%, P n = 5). These findings suggest that theinhibitory effect of PAF on proximal tubule function was not due to aninhibition of mitochondrial respiratory activity. Treating proximaltubules with 5 mM ouabain (Na -K -ATPaseinhibitor) reduced basal Q O 2 by 33 ± 5%( P n = 10) and abolished thestimulatory action of nystatin, confirming that the nystatin effect onQ O 2 was by increasingNa -K -ATPase activity. Conversely, ouabainreduced nystatin-stimulated proximal tubule Q O 2 by 58 ± 2% ( P n = 9)and abolished the inhibitory action of PAF. Together, these findingsindicate 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 tubularaction of PAF, we examined the ability of the phospholipid to inhibitnystatin-stimulated cellular transport in tubules preincubated with thePAF receptor antagonist BN-52021. As depicted in Fig. 4, we found thatthe inhibitory action of PAF on proximal tubule Q O 2 was abolished by 1 µM BN-52021. Thiseffect of BN-52021 appeared to be specifically related to blocking thePAF receptor, because it did not effect the inhibitory action of 1 pMANG IV on nystatin-stimulated proximal tubuleQ O 2 (ANG IV, 19.5 ± 1.9%, vs. ANGIV BN-52021, 18.2 ± 0.6%, n = 2 each), anANG IV response known to be mediated by the angiotensin AT 4 receptor ( 14 ).0 o- S- k! t7 r7 A; c, n8 p
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We then examined possible intracellular signaling mechanisms involvedin the inhibitory action of PAF on tubule Q O 2.The contribution of the nitric oxide-guanylate cyclase pathway wasassessed by preincubating proximal tubules with either 10 µMmethylene blue (guanylate cyclase inhibitor) or 100 µM L -NAME (nitric oxide synthase inhibitor). Curiously, weobserved that the increase in Q O 2 on nystatinadministration was quickly dissipated in tubules pretreated withmethylene blue only. However, both methylene blue and L -NAME treatments did not interfere with the inhibitoryaction of PAF on nystatin-stimulated proximal tubuleQ O 2 (Fig. 5 ). Incontrast, phospholipase A 2 (PLA 2 ) inhibitionwith 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 ofcytochrome- P -450 fatty acid -hydroxylase, data for 1 and10 µM were similar and combined) resulted in a 40-60% reductionin the effect of PAF on nystatin-stimulated proximal tubuleQ O 2 (Fig. 5 ). Basal andnystatin-stimulated Q O 2 were similar in alldrug-incubated tissues (the only exception was a depressed nystatinresponse in methylene blue-treated tissues) compared with untreatedcontrol tissue.* V% w9 p) n1 k% o3 F* f/ E

Fig. 5. Lack of effect of 10 µM metheylene blue (MB; guanylatecyclase inhibitor, n = 8) or 100 µM L -NAME (nitric oxide synthase inhibitor, n = 10) to alter the reduction in proximal tubuleQ O 2 induced by 1 nM PAF ( n = 12). Reduction in proximal tubule Q O 2 by 1 nMPAF ( n = 11) and its modification by 0.1-1 mMquinacrine (phospholipase A 2 inhibitor, n = 9), 50 µM SKF-525A (cytochrome- P -450 monooxygenaseinhibitor, n = 9), and 1-10 µM 17-octadecynoicacid (17-ODYA; cytochrome- P -450 fatty acid -hydroxylaseinhibitor, n = 14). * P) d2 X( j  q4 \
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DISCUSSION
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Early reports from our laboratory demonstrated that PAFadministered as a bolus ( 10 ) or infusion ( 11 )into the renal artery of anesthetized rats resulted in a decrease inrenal vascular resistance and reactivity, and we had speculated on theinvolvement of endothelium-derived relaxing factor (nitric oxide) inthese 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 ). Thepresent study also suggests that nitric oxide contributes to the renal vasodilatory and systemic hypotensive responses to intrarenal PAFinfusion in the rat.- `6 }5 W3 x  t. v1 N8 }; l: |

An intravenous infusion of AVP was used to raise systemic bloodpressure 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 forthe inability of intravenous pressor doses of AVP to decrease RBF inconscious rats, because renal denervation unmasked a vasopressin V 1 receptor-mediated fall in RBF ( 33 ). Ourfindings in anesthetized rats would not support this proposal, becausewe found no evidence of an AVP-mediated decrease in RBF underconditions in which the associated increase in blood pressure wasprevented from being transmitted to the denervated kidney. Therefore,we conclude that it is unlikely that the renal vasodilation induced byPAF 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 highpharmacological doses of AVP ( 11 ). In previous studies, wecould only show weak and transient renal vasodilatory responses tononhypotensive intrarenal infusion rates of PAF ( 11 ).However, the experimental setup employed in the present study(preventing the PAF-induced systemic hypotension and associatedincrease in sympathetic outflow from being transmitted to thedenervated kidney) allowed us to reveal a strong and sustained kidneyvasodilatory response to intrarenal PAF infusions in normal rats in vivo.0 S: m  |4 Q1 s0 _+ U
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To our knowledge, the present results are the first to demonstrate thatPAF infusion into the rat renal artery can be associated with asignificant diuresis, natriuresis, and kaliuresis, the magnitude ofwhich was attenuated by nitric oxide synthase inhibition. However,opposing our in vivo findings is the report that the intrarenalinfusion of PAF into anesthetized rats resulted in a decrease inurinary water excretion rate and U Na V that was largely dueto PAF-induced falls in RBF and glomerular filtration rate ( 42 ). Although we cannot readily explain these hemodynamicdiscrepancies, it should be noted that a number of investigators havedemonstrated that PAF can possess vasodilatory properties in the kidney(10, 17; see also citations in Ref. 11 ) andinhibit solute reabsorptive processes by acting at the level of thenephron ( 2, 5, 19, 29 ). In the present study, we regulatedrenal arterial blood pressure such that the AVP-induced rise insystemic blood pressure was not transmitted to the denervated kidneyand found that urinary water and solute excretion remained unchanged.The actions of AVP in the kidney have been reported to range from adecrease to an increase in urinary water excretion rate andU Na V, as well as any conceivable combination of urinaryexcretory responses between these two extremes ( 3, 8, 9, 18, 20 ). This highlights the complex and multifactorial nature ofAVP actions in the kidney to regulate urinary water and soluteexcretion, including integrated effects on blood pressure,sympathoinhibition, renal hemodynamics, and tubular function ( 3, 8, 9, 18, 20 ). Although one would expect the direct stimulatoryactions of AVP on tubular transport processes to result in a decrease in urinary water excretion rate and U Na V ( 3 ),investigators have also reported that nonpressor doses of AVP infusedinto the kidney can be associated with no change in both renalhemodynamics and urinary water excretion rate and/or U Na V( 8, 20 ). Consequently, we conclude that the subsequentintrarenal infusion of PAF was likely responsible for the observedincrease in urinary water and solute excretion in the anesthetized rat.In addition to a significant role for nitric oxide in the glomerularand vascular actions of PAF in the kidney ( 13, 17, 24 ),the renal nitric oxide pathway is also known to be natriuretic byinfluencing several kidney systems, including the cortical andmedullary microcirculation, interstitial hydrostatic pressure, andtubular transport processes ( 21, 25, 37 ). In the presentstudy, blockade of nitric oxide synthesis was found to dramaticallyattenuate the ability of PAF to elicit a diuresis, natriuresis, andkaliuresis and suggests either a direct or indirect role of nitricoxide in these urinary water and electrolyte responses.0 W6 b8 O: N1 C& L7 y

Because our in vivo results do not allow us to dissociate the urinaryexcretory responses from the PAF-mediated renal hemodynamic effects, weexamined the direct actions of PAF on isolated rat proximal tubules,because this nephron segment is a major site for the reabsorption ofsodium and water and contains abundant PAF receptor mRNA( 1 ). No information is available on the direct actions ofPAF on this segment of the nephron. We found that PAF inhibitedproximal tubule Q O 2 without influencingmitochondrial uncoupled Q O 2 rates, suggesting areduction in energy-dependent transcellular solute transport. Proximaltubules were treated with nystatin (sodium ionophore that functionallybypasses the rate-limiting step of sodium entry into the cell),allowing an indirect assessment of sodium efflux from the cell via thebasolateral Na -K -ATPase pump( 23 ). Nystatin treatment increased the sensitivity of theproximal tubules to the inhibitory actions of PAF, suggesting that thelipid may act to reduce basolateralNa -K -ATPase pump activity. This conclusion issupported by the observation that ouabain, anNa -K -ATPase inhibitor, prevented theinhibitory effect of PAF on nystatin-stimulated proximal tubuleQ O 2, as well as several reports that PAF caninhibit membrane Na -K -ATPase activity in anumber of cell types ( 4, 16 ). In addition, endogenous PAFwas found to contribute to the decrease in renal cortexNa -K -ATPase activity after reperfusion of theischemic rat small intestine, an effect that could be blockedby the PAF receptor antagonist BN-52021 ( 39 ). Our resultsobtained with D -PAF, lyso-PAF, and PAF in the presence ofBN-52021 would also be consistent with a PAF receptor mediating thebiological response of PAF in isolated rat proximal tubules.1 t4 F  W  k( n% P
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The dilatory action of PAF on the renal vasculature is largelydependent on the nitric oxide pathway (present study and Ref. 17 ), and others have shown that activation of guanylatecyclase and the subsequent rise in cGMP mediate many actions of nitric oxide in the body ( 26 ). We found that nitric oxideblockade with L -NAME attenuated the natriuretic anddiuretic response to intrarenal PAF infusion in the rat in vivo. Thisreduced urinary excretory response could be due to L -NAMEpreventing PAF-induced renal vasodilation and/or reducing anatriuretic/diuretic action of PAF at the level of the nephron. Bothnitric oxide and cGMP can be produced in the proximal tubule, with bothcapable of inhibiting proximal tubuleNa -K -ATPase activity ( 21 ).However, we found that methylene blue and L -NAME atconcentrations known to inhibit tubular guanylate cyclase and nitricoxide synthase activity, respectively, did not interfere with theinhibitory actions of PAF on rat proximal transport processes. Thiswould seem to rule out nitric oxide as a candidate for mediating thedirect inhibitory effect of PAF on proximal tubule solute reabsorptivefunction. Although it is presently unknown whether PAF can stimulatenitric oxide biosynthesis and/or guanylate cyclase activity in theproximal tubule, the phospholipid can stimulate cGMP in glomerularcells when coincubated with endothelial cells ( 24 ) andinhibit solute reabsorptive function of the medullary thick ascendinglimb (mTAL) via a cGMP-dependent pathway ( 29 ). Our resultsdo not rule out the possibility that PAF-stimulated nitric oxide/cGMPsynthesis from the renal microcirculation and other nonproximal tubulesources may inhibit proximal tubule solute reabsorptive processes invivo ( 21 ). Further downstream of the proximal tubule,investigators have demonstrated that PAF can reduce the ability ofvasopressin to increase transepithelial resistance (a measure of activesolute reabsorption) in cultured rat inner medullary collecting ductcells ( 19 ) or microperfused rabbit cortical collectingducts ( 5 ). This would likely result in a natriuresis anddiuresis in vivo, because this terminal segment of the nephron isimportant in regulating the final amount of sodium and water that isexcreted in the urine.

The arachidonic acid pathway appeared to be of major importance in thedirect actions of PAF in the rat proximal tubule. Inhibitors ofPLA 2 and cytochrome- P -450 monooxygenase wereable to significantly attenuate the inhibitory actions of PAF onproximal tubule Q O 2. This is similar to theproposed pathway by which a number of renal hormones inhibit proximaltubule Na -K -ATPase activity( 34 ). PAF is a potent activator of PLA 2,resulting in the formation of arachidonic acid ( 15, 28, 36 ). Processing of arachidonic acid bycytochrome- P -450 monooxygenase can lead to the formation ofHETEs and epoxyeicosatrienoic acids that can reduce the transcellularmovement of sodium across the proximal tubule; e.g., 20-HETE inhibitsbasolateral Na -K -ATPase activity through aPKC-dependent phosphorylation of the -subunit of ATPase, whereasepoxyeicosatrienoic acids are natriuretic perhaps by inhibiting thetranslocation of the Na -H antiporter to theapical membrane ( 34 ). Although PLA 2 andcytochrome- P -450 monooxygenase products do not appear to beresponsible for the inhibitory effect of PAF on solute transportprocesses in the mouse mTAL ( 2 ), others have implicatedthe cytochrome- P -450 monooxygenase pathway in the cellularbiological actions of PAF in the rat hindlimb ( 38 ) andhuman neutrophil ( 27 ). Our results would suggest that onedirect action of PAF on the rat proximal tubule is to decrease sodiumtransport reabsorption via a PLA 2 andcytochrome- P -450 monooxygenase pathway and may reflect the fact that intracellular signaling systems activated by PAF in thenephron are site specific. However, our results did not reveal a majorcontribution of nonnitric oxide renal tubule pathways in the renalexcretory responses to intrarenal PAF infusion in nitric oxide synthaseinhibitor-treated rats in vivo. This is probably related to the factthat infusing PAF into the renal vascular compartment greatly limitsits access to renal tubules ( 30 ). PAF is not only acirculating lipid but is also synthesized by glomerular cells, renalmedullary interstitial cells, and inflammatory cells infiltrating thekidney that would allow both greater access and higher concentrationsof PAF at renal tubule sites. Presumably, this intrarenalgeneration/release of PAF would allow tubule PAF receptor systems tohave a greater role in regulating renal excretory function. On theother hand, we cannot exclude the possibility that the multiple andcomplex changes in renal function induced by nitric oxide synthaseinhibition may mask/attenuate the contribution of the renal tubules tothe PAF-induced diuresis, natriuresis, and kaliuresis.% \% i$ B3 N% _6 F5 l! p. ?
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In conclusion, a number of factors may potentially contribute to thePAF-induced increase in urinary water and electrolyte excretionobserved in vivo and include 1 ) PAF-induced increase inkidney blood flow that was secondarily mediated by nitric oxide, 2 ) perhaps PAF-stimulated nitric oxide synthesis from anonproximal tubule source leading to a reduction in proximal tubulesodium reabsorption, 3 ) PAF activation of proximal tubulePLA 2 and cytochrome- P -450 monooxygenase pathwaysto attenuate sodium reabsorption processes, and 4 )PAF-mediated inhibition of solute reabsorption in the mTAL andcollecting ducts.
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ACKNOWLEDGEMENTS
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This work was partly supported by an RJR-Leon Golberg ResearchAward in Pharmacology and Toxicology.
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