Evaluation of the micropuncture determination of single nephron filtration fraction

Transcription

1 Kidney Interntionl, Vol. 25 (1984), pp Evlution of the micropuncture determintion of single nephron filtrtion frction PETER K. JENSEN, HANNE BLAEHR, nd KENNETH STEVEN Medicl Physiology Deprtment A, The Pnum Institute, Copenhgen, Denmrk Evlution of the micropuncture determintion of single nephron filtrtion frction. Spontneous nd spirted blood smples were collected from the welling points of surfce efferent rterioles in the rt kidney. The mount of tubulr rebsorbte in ech smple ws clculted from mesurements of the inulin concentrtion in the smple nd systemic rteril plsm. Both spontneous nd spirted smples contined substntil mounts of tubulr rebsorbte (4% 1% nd 11% 1%, respectively). Accordingly, the single nephron filtrtion frction vlues derived from mesurements of lbumin concentrtions lone were significntly lower thn those clculted considering the ddition of the mount of tubulr rebsorbte in ech smple (.26.1 vs..3.1 nd.25.1 vs for the spontneous nd spirted smples, respectively). Incresing the rte of blood collection from the welling point by spirtion ws ssocited with significnt decrese in the hydrulic pressure in the sme vessel. The efferent rteriolr blood smples were therefore probbly contminted with tubulr rebsorbte s result of retrogrde flow from the peritubulr cpillries. It ws confirmed tht single nephron filtrtion frctions derived from hemtocrit mesurements were significntly higher thn those bsed on lbumin concentrtion mesurements, supporting tht the hemtocrit of fferent rteriolr blood of the superficil nephrons is higher thn tht of systemic rteril blood. Evlution de I determintion pr microponction de I frction de filtrtion néphromque individuelle. Des échntillons de sng spontnés ou pr spirtion ont étd collectés prtir de points d'emergence d'rtérioles efférentes superficielles dns du rein de rt. L quntite de rébsorbt tubulire dns chque échntillon été clculée prtir de mesures de l concentrtion d'inuline dns l'échntillon et le plsm rtériel systémique. Les Cchntillons spontnés et spires contenient des quntités substntielles de rébsorbt tubulire (4% 1% et 11% 1%, respectivement). De I sorte, les vleurs de frction de filtrtion néphronique individuelle obtenues prtir des seules mesures de concentrtions d'lbumine étient significtivement plus fibles que celles clculées en prennt en compte en plus I quntite de rébsorbt tubulire dns chque échntillon (,26,1 contre,3,1 et,25,1 contre,34,1 dns les échntillons spontnés et spires, respectivement). L'ugmenttion du debit de collection sngum prtir du point d'emergence pr spirtion étit ssociée une diminution significtive de I pression hydrulique dns le méme visseu. Les dchntillons snguins rtériolires efférents étient de I sorte probblement contminés vec du rébsorbt tubulire resultnt d'un flux retrogrde prtir des cpillires peritubulires. Ii été confirmé que les frctions de filtrtion nephronique individuelle obtenues vec des mesures de l'hémtocrite étient significtivement plus fortes que celles reposnt sur des mesures de concentrtion d'lbumine, cc qui indique que l'hémtocrite du sng rtériolire fférent des néphrons superficiels est plus Clevé que celui du sng rtériel systemique. Received for publiction December 16, 1982 nd in revised form My 2, by the Interntionl Society of Nephrology Glomerulr filtrtion dynmics, to our knowledge, hve been investigted minly in superficil nephrons [1]. Much ttention in these investigtions hs been focused on the phenomenon of filtrtion equilibrium which occurs when the oncotic pressure of the plsm proteins t the end of the glomerulr cpillry reches the sme vlue s the hydrulic pressure difference cross the ultrfiltrtion membrne [1, 2]. This observtion is bsed on mesurements of the protein concentrtion in plsm obtined from the welling point of the efferent rteriole on the renl surfce. Experiments hve shown tht the inulin concentrtion in efferent plsm is lower thn the corresponding systemic vlue which hs been tken s evidence for the uptke of tubulr rebsorbte long the efferent rteriole [3]. Collection of blood from the efferent rteriole will reduce the hydrulic pressure t the collection site which could cuse retrogrde flow of blood from the peritubulr cpillries. This would mimic uptke of tubulr rebsorbte in the efferent rteriole nd reduce the protein concentrtion in the collected plsm. Consequently, the glomerulr cpillry filtrtion frction is underestimted nd the vrince is incresed [4]. These possible sources of error should be considered in experiments on the occurrence of filtrtion equilibrium of the glomerulr ultrfiltrtion process [1, 2]. The correct filtrtion frction cn be determined experimentlly using two trcers simultneously [5]. One trcer should be vsculr impermeble nd the second freely filtrble cross the glomerulr cpillries nd subsequently not be trnsported cross the tubulr epithelium. Mesurement of the ctivity of the second trcer in the collected efferent blood then mkes it possible to clculte the mount of tubulr fluid rebsorbed in the postglomerulr microcircultion. Knowing this, the ctivity of the vsculr impermeble trcer t the end of the glomerulr cpillries cn be clculted from the mesured welling point ctivity nd the correct filtrtion frction cn thus be determined. The present study ws mde to determine the filtrtion frction of single superficil rt nephrons by collecting blood from the efferent rteriolr welling points on the renl surfce using two simultneous trcers. The effect of the rte of efferent blood collection on the hydrulic pressure in the sme welling point ws studied by mens of volume trnsducer nd servonulling pressure mesuring device [6]. The effect of collection rte on the mount of tubulr rebsorbte dded to the collected plsm ws investigted. In ddition, the possibility of intrrenl sequestrtion of erythrocytes [7] ws studied by 486

2 Single nephron filtrtion frction 487 comprison of the filtrtion frctions determined both from the of FF using Eq. (6) will result in n underestimte with the hemtocrit nd protein concentrtion in the smples. reltive error, : Theory. Mss conservtion of trcer long the glomerulr cpillries nd the efferent rteriole requires tht: = Eq. (6) = Cw (I) 1 (J) <CF (II) (7) Eq. (5) CF CA CA QA = C (QA SNGFR + ABS) + CF SNGFR (1) Using erythrocytes s nonfiltrble trcer [11 gives in where C denotes trcer concentrtions nd subscripts F, A, nd nlogy to Eq. (3): W refer to glomerulr ultrfiltrte, systemic rteril plsm, nd efferent plsm from the welling point, respectively. QA is the FF = 1 1 l/hctw 1 1/HctA rte of fferent rteriolr plsm flow. SNGFR is the rte of (8) single nephron glomerulr ultrfiltrtion, nd ABS is the rte of where Hct is the erythrocyte volume frction. The pprent tubulr fluid rebsorption in the postglomerulr microcirculhemtocrit of the blood entering the fferent rteriole cn be tion. No net trnsport of trcer is ssumed to occur cross the clculted by rerrnging this eqution nd inserting the vlue postglomerulr vsculr wlls or the tubulr epithelium. Diviof FF clculted by Eq. (3): sion by QA C nd rerrngement of Eq. (1) gives: AF = FF (1 CF/Cw) (1 CA/Cw) 1 Hct 1/Hctw A* = (2) (i 1 FF ) (9) where the sterisk indictes the clculted vlue of fferent where FF is the frction of fferent rteriolr plsm flow tht is rteriolr hemtocrit. This eqution is identicl to the equtions ultrfiltered in the glomerulus nd AF is the frction rebsorbed given by Brenner nd Gll [1] nd by Jckson nd Oken [4] in the postglomerulr microcircultion. prt from printer's error [4]. If no intrrenl sequestrtion of Provided both AF nd CF re zero Eq. (2) reduces to the erythrocytes occurs, the fferent rteriolr hemtocrit predict- Bresler eqution [8]: ed from Eq. (9) should be equl to the systemic rteril FF = 1 hemtocrit. CA/Cw (3) Methods which is usully employed for determintion of FF using nonfiltrble trcers [1, 2, 4]. A breeding nucleus of Wistr rts possessing surfce glomer- Provided AF is zero nd the trcer is freely ultrfiltrble uli ws obtined from Munich (strin WU, Ivnovs, Kisleg im CF = CA/fw rerrnging Eq. (2) gives the concentrtion rtio: Algu, Federl Republic of Germny). From the first descendent genertion 38 mle rts weighing between 195 nd 482 g C 1 FF/fw (men weight, 35 g) were nesthetized with mctin (11 to 12 CA 1 FF < 1 (4) mg/kg body wt, i.p.) nd prepred for micropuncture of the left kidney s described previously [11]. Briefly, ech niml ws where fw is the plsm wter frction usully mesured by plced on heting pd nd the trche ws cnnulted. refrctometry [9]. Ctheters were plced in the bldder, left ureter, femorl If AF is not the zero clcultion of the two unknown vribles rtery, nd vein. The kidney ws immobilized in plstic cup in Eq. (2), AF nd FF, requires two trcers (I nd II). nd superfused by minerl oil t 37 C. Heprinized plsm Eliminting AF from Eq. (2) then renders: obtined from littermtes ws infused (1 ml/hr/kg body wt for 45 mm during the preprtion nd 1.5 mi/hr/kg body wt for the C reminder of the experiment) s described by Ichikw et l FF Cw [12]. CF After preprtion ech niml received 6 pci/kg body wt of (I) (j '311-lbumin (humn serum lbumin, IK21S, Kjeller A/S, Oslo, Norwy) nd 2 U/kg body wt of heprin intrvenously The error in predicting FF is minimized if (I) is freely filtrble dministered in volume of.2 to.5 ml of physiologicl sline. nd (II) completely restricted by the glomerulr membrne. Simultneously, infusion of 5 pci/hr of '4C-inulin (CFA 399, Accordingly, the substitution of CF(I) = CA(I)/fw nd CF(II) = Amershm Rdiochemicl Center, United Kingdom) in physioin Eq. (5) results in: logicl sline ws initited t rte of 1.2 mi/hr. Experiments were strted fter 1-hr equilibrtion period. FF = fw C(I) CA(II) " Proximl tubulr fluid smples were obtined t the pre- (1 (6) C(JI) CA(I)) existing free flow pressure by collection into oil-filled micropipettes (8 to 1 tm I.D.) s described previously [11]. To collect which is identicl to the expression given by Nissen [5]. Using blood from the efferent rteriole, lrge peritubulr cpillry rdioctive trcers, the concentrtions in Eq. (6) cn be substi- ws punctured by siliconized pipette (12 to 14 jm I.D.) filled tuted by the totl ctivities thus eliminting the error in mesur- with minerl oil. The tip of the pipette ws then dvnced into ing smll volumes, the welling point of the efferent rteriole [1] nd the intrvs- If trcer (II) is not completely restricted from permetion culr locliztion ws verified by injecting few drops of through the glomerulr membrne, CF(II) >, the clcultion minerl oil. Thirty-nine spontneous smples were initited by

3 488 Jensen et l single brief spirtion followed by spontneous flow of.17 for the inulin nd lbumin trcers, respectively. blood into the pipette. Twenty-nine spirted smples were The equtions nd the procedures were tested by ultrfilterobtined by pplying continuous or intermittent suction to the ing 5 ml of rteril plsm (CF 25 centriflo membrne cone, collection system throughout the collection period. At the end Amicon, Lexington, Msschusetts). The filtrtion frction of ech collection slight mount of the collected blood ws clculted by Eq. (3) using the protein concentrtions (refrcreinjected into the welling point to verify the intrvsculr tometry) ws.46. The filtrtion frction clculted by Eq. (5) locliztion of the pipette tip. The smple ws discrded if using the isotope counts in 5 ILl smples ws.47.1 (SEM, tubulr or n interstitil fistul ws found. To prevent subcp- N = 5). After dding 1% of wter to the remnnt plsm, the sulr bleeding, the pipette ws then withdrwn very slowly nd filtrtion frction clculted with Eq. (5) ws unchnged t.47 crefully to prevent fluid movements into the tip. Systemic.2 (SEM, N = 5) nd the bsorption frction clculted with rteril blood smples (75 ILl) were obtined t regulr intervls Eq. (2) ws.9.2 (SEM, N = 5). from the femorl ctheter, To clculte the low ctivity of 1311 in tubulr fluid, the The micropipettes were centrifuged (tip down) inside n oil- counts of ll the bckground nd tubulr smples of ech niml filled hemtocrit cpillry closed by wx (Sigillum, Dnsk were pooled in two groups. The difference in ctivity between Lbortorieudstyr, Copenhgen, Denmrk) to sel the tip open- the groups were corrected for the decy between successive ing of the pipette during centrifugtion. The volume frction of countings. The tubulr fluid ctivity ws then tken to be the erythrocytes in ech microsmple ws then determined directly men of these differences; the trcer concentrtion ws clcuin the collection pipette by mesuring the length of the erythro- lted from the totl mount of tubulr fluid collected in ech cyte column in the shnk plus one third of the length in the cone niml. The rnge of vrition coefficients ws 3 to 484% with of the pipette. This method voids trnsference of the smple to medin vlue of 28%. microhemtocrit cpillry. It ws vlidted by 23 microsm- In nine seprte experiments the rte of collection of efferent pies pired with mcrosmples (75 d) in stndrd hemtocrit blood ws mesured continuously by volume trnsducer [6]. tubes (red tip, Dnsk Lbortorieudstyr). The men difference This ws ssembled by connecting the trnsducer to micropiof the pirs ws only.1.3 (sem). The coefficients of pette (I.D., 14 ILm) filled by mercury. Minerl oil ws then vrition were.1 to.9% nd 1.5 to 4,2% for the mcro- nd drwn into the tip until the mercury column entered the the micromethod, respectively, within the rnge of hemtocrits constnt bore of the trnsducer [6]. A welling point ws from.42 to.61. punctured nd spontneous collection of blood into the Ech pipette ws cut bove the plsm-oil interphse fter pipette ws initited s described bove. The pressure t the hemtocrit ws determined; the plsm ws trnsferred to cup sme loction ws mesured by servonulling pressure mesur- (Teflon ) filled by silicone oil for volume mesurements [13]. ing system (Model 4A, Instrumenttion for Physiology nd Smples with volume less thn 1 ni were discrded, To Medicine, Sn Diego, Cliforni) using 2-ILm pipettes filled by 1 mesure 1311-lbumin ctivity, the remining smples were M NCl, 1% lissmine green [6, lii. The pressure pipette ws trnsferred to counting vils (3 ml polyethylene vils, Lumc, introduced into the sme welling point very crefully through Schlsberg, The Netherlnds) contining.25 ml of distilled one of the remining first order cpillries nd stble pressure wter nd counted twice for 6 mm or 1, counts in gmm trce ws estblished. The rte of blood collection ws then spectrometer (Auto gmm scintilltion spectrometer, Pckrd chnged by ltering the pressure in the spirtion system model 5221, Pckrd Instruments Co., Downers Grove, Illi- connected to the volume trnsducer. Instntneous chnges in nois). No correction for the decy of 131J ctivity (hlf-life 8.4 the pressure in the welling point followed ltertions of the dys) during counting ws performed becuse ech efferent collection rte. The men pressure ws tken when the collecrteriolr smple ws brcketed by corresponding systemic tion rte hd remined constnt during smpling of t lest 25 nl plsm smple in duplicte (5 gd). To determine the ctivity of of efferent rteriolr blood. The volume trnsducer ws cli- '4C-inuiin, the vils were stored for pproximtely 8 weeks t brted fter ech experiment by 5-jil microsyringe (Hmil- 2 C to let the ctivity of 1311 decy below 1% of the inulin ton, Bonduz, Switzerlnd) mounted on pump (model 341, ctivity. After storge the vils were llowed to stnd overnight Sge Instruments Inc., Cmbridge, Msschusetts). t room temperture nd 2.5 ml of scintilltion fluid (Lumgel, Sttisticl nlyses were mde with the pired or unpired Lumc) ws then dded to ech smple. The ctivity ws Student's t test. The results re given s the mens SEM. The determined in bet counter (Tri-crb liquid scintilltion spec- sources of vrition were determined by nlysis of vrince trometer, Pckrd model 245). The series contined stndrds using one-wy rndom effects model [14]. The vrince of '31I-lbumin to correct for bet ctivity originting from between nimls (rb2) ws clculted from the men sum iodine, squres within (S2) nd between nimls (SB2): To exclude preferentil dsorption of one isotope to the (SB 2 2 sw ) (k 1) N pipette wll, the procedures were vlidted by 18 pirs of N (1) mcro- (5 d) nd microsmples (men 273 ni, rnge 65 to 581 nl) tken from rteril plsm nd subsequently treted in mnner identicl to the experimentl smples. The men rtio where k is the number of nimls, n1 is the number of smples in between the totl ctivities of the two isotopes in the pired the ith niml nd N is the totl number of smples [14]. smples ws 1. (vrince =.29, N = 18) which is not significntly different from unity. This rtio is equivlent to the rtio ppering in Eq. (6). The vrinces for the concentrtion rtios between the micro- nd mcrosmples were.22 nd Results Figure 1 gives the results of nine experiments showing tht incresing the rte of blood collection from the welling point of

4 Single nephron filtrtion frction 489 E E Rte of collection, l/mm Fig. 1. Pressure in the welling point (Pw) plotted ginst the rte of collection ofbloodfrom the sme vessel. Successive vlues determined by volume trnsducer from ech individul welling point re connected. the efferent rteriole by pplying suction to the collection system reduced the hydrulic pressure in the sme vessel. The men superficil single nephron filtrtion frction of the pooled spontneous nd spirted smples ws clculted t.25.1 (N = 68) with Eq. (3) using only the concentrtions of iodinted lbumin. The internephron heterogeneity ws the min source of vrition since the vrince within nimls ws.39 while the vrince between nimls ws.1. Hence, only 21% of the totl vrince ws due to interniml heterogeneity. In both the spontneous (N = 39) nd the spirted (N = 29) smple groups, the men rtio between the inulin concentrtions in welling point plsm nd systemic rteril plsm ws significntly lower thn tht predicted from Eq. (4) (Tble 1). The men pired difference ws lrger for the spirted thn for the spontneous smple group being.11.1 (P <.1) nd.4.1 (P <.1), respectively. Accordingly, both groups of smples contined significnt mount of inulin-free tubulr rebsorbte. To determine the correct filtrtion frction therefore requires the use of Eq. (6) tht includes the mount of tubulr rebsorbte in the smple. The corrected filtrtion frctions re shown in Figure 2 nd Tble 1. The vrince of the corrected filtrtion frction within nimls ws.48 wheres the vrince between nimls ws.5; thus, only 1% of the totl vrince ws due to the vrition between nimls. The concentrtion rtio of the iodine trcer in proximl tubulr fluid to systemic rteril plsm ws Clcultion of the single nephron filtrtion frction with Eq. (5) tkes ccount of this smll but highly significnt mount of trcer ultrfiltered in the glomerulus (Tble 1). The men pired difference between the vlues clculted by Eqs. (5) nd (6) ws.1 (N = 68, P <.1), so the men reltive only.5 error by using Eq. (6) insted of Eq. (5) ws clculted (Eq. (7)) t only 1.5.2%. Figure 3 shows tht the single nephron filtrtion frction vlues derived from hemtocrit mesurements (Eq. (8)) were significntly higher thn those derived from lbumin concentrtion mesurements. The men pired difference between the vlues clculted with Eqs. (8) nd (3) ws.1.1 (N 68, P <.1). Knowing the filtrtion frction derived from lbumin concentrtion mesurements (Eq. (3)) nd the efferent rteriolr hemtocrit, the fferent rteriolr hemtocrit ws clculted with Eq. (9). The clculted men vlue ws.37.6 (N = 68, P <.1) higher thn the systemic rteril hemtocrit of This pired difference ws similr in the spontneous nd spirted smple groups being.36.7 nd.38.9, respectively. Discussion Recent studies hve shown tht the concentrtion of severl substnces in plsm collected from the efferent rteriole differs from the corresponding concentrtions in systemic rteril plsm [3]. This observtion hs been tken s evidence for uptke of tubulr rebsorbte long the course of the efferent rteriole. The conventionl method of determintion of single nephron filtrtion frction by mesurements of the protein concentrtion in efferent rteriolr plsm [81 is not vlid if the proteins re diluted by tubulr rebsorbte t the point of collection of postglomerulr blood [31. A method hs been designed previously to circumvent this problem by using two trcers simultneously for determintion of both the filtrtion frction nd the frction of fluid rebsorbed in the postglomerulr microcircultion [5]. In the glomerulr cpillries one trcer (lbumin) should be restricted completely from pssge cross the filtrtion membrne, wheres the second trcer (inulin) should be freely filtrble. The inulin trcer monitors the rebsorption tht occurs pst the glomerulus so tht the concentrtion of lbumin t the glomerulr cpillry end cn be corrected ccordingly. Idelly, the two trcers should not be trnsported cross the postglomerulr vsculr membrnes or the tubulr epithelium. The requirement of mss conservtion of the trcers is fulfilled in the renl circultion since only negligible mounts of trcers re lost vi the renl lymphtic vessels [15]. Only negligible frction of the lbumin trcer is filtered in the glomerulus so lmost the sme filtrtion frction is clculted with Eq. (6) using mesurements of the ctivity rtios s with the more correct Eq. (5) requiring mesurements of the bsolute concentrtions of ech trcer. The in vitro tests show tht the present method of determining the filtrtion frction is ccurte nd tht the recovery by processing the microsmples is 1%. Its min disdvntge is the dely in the counting procedure. To overcome this dely two iodine gmm emitters could be used with iothlmte s the filtrble trcer. Helth resons exclude this possibility becuse of the lrge mounts of trcers needed for micropuncture experiments. Alterntively, high energy protein bound bet emitter in combintion with 3H-inulin would be preferble. To our knowledge, such trcer is not vilble. The filtrtion frction my lso be determined by simultneous mesurements of single nephron clernces of '4C-inulin nd 3H-PAH by quntittive collection of distl tubulr fluid. This method requires knowledge of the single nephron extrction frction of PAH which cnnot be determined simultneously with rdioctive trcers. After correction of the filtrtion frction, the min source of vrition of the filtrtion frction is cused by vrition within the nephron popultion in given niml. This vrince com-

5 49 Jensen Ct! Tble 1. Men vlues Mode of collection Spontneous Aspirtion P vlue SEM ofthe welling point plsm to rteril plsm inulin concentrtion rtio (Cw/CA) ssuming no ddition of tubulr rebsorbte (Eq. 4, Theory section) nd the experimentl observtion (OBS) N CW/CA Eq. (4) NS CW/CA OBS <.1 AF Eq. (2) <.1 FF Eq. (3) NS FF Eq. (5).3.34 <.2 FF Eq. (6) Single nephron rebsorption frction (AF) nd glomerulr filtrtion frctions (FF) were clculted solely from the lbumin concentrtion rtio (Eq. 3, Theory section), from lbumin nd inulin concentrtions (Eq. 5, Theory section), nd from the rtio of lbumin nd inulin ctivities (Eq. 6, Theory section) < ' C.) C.) C C.) E O FF uncorrected Fig. 2. Filtrtion frction clculted from Eq. (6) (FF corrected) plotted ginst the filtrtion frction clculted from Eq. (3) (FF uncorrected). The line of identity is indicted. Circles nd tringles show vlues for spontneous nd spirted smples, respectively FF protein Fig. 3. Filtrtion frction clculted from Eq. (8) (FF hemtocrit) plotted ginst the filtrtion frction clculted from Eq. (3) (FF protein). The line of identity is indicted. Circles nd tringles show vlues for spontneous nd spirted smples, respectively. prises biologicl internephron heterogeneity nd methodologicl vribility. It is not possible to seprte clerly between these two sources of vrince in the present study s only single punctures nd single determintions were performed on the experimentl microsmples. The in vitro study designed to ssess methodologicl vribility ssocited with the processing of the microsmples shows tht bout hlf of the vrince within nimls cn be scribed to the methods. The experimentl results confirm tht the inulin concentrtion in efferent rteriolr plsm collected from the welling point hs lower inulin concentrtion thn expected from the corresponding vlue in systemic rteril plsm [3] even fter the correction for incresed contents of solids in efferent plsm (Eq. (4)) hs been mde. This observtion indictes the ddition of inulin-free tubulr rebsorbte to the smple which could be the result of tubulr rebsorbte uptke through the efferent rteriolr wll or be cused by retrogrde flow from the peritubulr cpillry network. The former explntion ppers very unlikely since the mximl mount of fluid uptke long the efferent rteriole cn be estimted from the product of the trnsvsculr driving pressures, efferent rteriolr surfce re, nd hydrulic permebility s follows: The oncotic pressure minus the hydrulic pressure difference mximlly would be bout 25 mm Hg [16], the efferent rteriolr surfce.16 x l- cm2 (ssuming dimeter of 2 m nd length of 25 sm), nd the hydrulic permebility probbly not exceeding the vlue found for the peritubulr cpillry of 1'o cm/sec/mm Hg [17]. The totl mount of rebsorbte would ccordingly be 4 p1/sec which is severl orders of mgnitude below the plsm flow through the efferent rteriole of bout 2 ni/sec [1, 2, 1, 11, 16]. The second explntion of retrogrde flow from the peritubulr cpillries seems more likely since model simultion of the glomerulus [18] hs shown tht decrese in the hydrulic pressure in the efferent rteriole hs only smll effect on the rte of glomerulr blood flow. Accordingly, the very lrge collection rtes ssocited with spirted smples (Fig. I) re

6 Single nephron filtrtion frction 491 most likely cused by retrogrde flow from djcent nephrons due to reversl of the hydrulic pressure grdient long the communicting network of peritubulr cpillries. This explntion ccounts for the lower inulin concentrtion in spirted smples due to the ddition of inulin-free tubulr rebsorbte to the peritubulr cpillry blood. Also, in smples collected spontneously, there is slight but significnt ltertion of inulin concentrtion, possibly due to retrogrde flow tht would go unnoticed visully if it occurred in brnches from the efferent rteriole beneth the renl surfce [1]. This explntion ppers unlikely since it requires reversl of the pressure grdient tht should lso cuse retrogrde flow in the superficil cpillries. Alterntively, it is possible tht the puncture of the efferent rteriole cuses locl increse of the hydrulic permebility. The observtion of incresed corrected filtrtion frction in the group of spirted smples is surprising since model clcultions [18] show tht decrese of the peritubulr cpillry pressure will cuse decrese of the filtrtion frction minly s result of decresed glomerulr cpillry pressure. It is conceivble tht the collpse of the cpillries nd efferent rteriole observed during spirtion, which grees with previous observtions of the peritubulr cpillries being highly elstic [19], my result in n increse of the efferent resistnce. This would prevent the glomerulr cpillry pressure from flling. The decrese of peritubulr cpillry pressure increses the rte of tubulr rebsorption [16], which hs been shown in recent model study [2] to decrese intrtubulr pressure nd consequently increse the filtrtion frction. The hemtocrit mesurements hve been used previously to determine the filtrtion frction of superficil rt nephrons [4, 1]. The present experiments gree with these studies showing tht the filtrtion frction vlues bsed on hemtocrit mesurements cn exceed those derived from protein concentrtion mesurements s observed in isovolemic plsm-exchnged rts [11 nd hydropenic rts [4]. This observtion cn be explined by plsm skimming in the deep prt of the interlobulr rtery [7] cusing n incresed hemtocrit of blood flowing to the fferent rterioles of superficil nephrons. Plsm skimming does not occur t the tip of the collecting pipette since the hemtocrit vlues mesured by the micro- nd the mcromethod were identicl, which grees with previous study [1]. It is unlikely tht sequestrtion of erythrocytes occurs in the postglomerulr microcircultion by preferentil collection of erythrocytes following the fstest flow [21] since the sme difference between pprent fferent rteriolr hemtocrit nd systemic rteril hemtocrit ws obtined by different collection rtes. Accordingly, the sequestrtion of erythrocytes must occur by skimming the plsm long the interlobulr rtery. This conclusion is substntited by observtions of smller number of erythrocytes in blood collected from vse recte in the exposed ppill thn in the corresponding systemic blood [22]. In summry, our results show tht mesurements of the filtrtion frction of superficil nephrons re subject to rtifcts which must be considered in studies on the occurrence of filtrtion equilibrium. Acknowledgment The experiments were supported by Lndsforeningen for Sukkersyge nd the Mdsen Foundtion. N. Rsmussen nd B. Avd provided technicl ssistnce. Reprint requests to Dr. P. K. Jensen, Medicl Physiology Deprtment A, The Pnum Institute, Blegdmsvej3, DK-22 Copenhgen N. Denmrk References I. BRENNER BM, TROY JL, DAUGHARTY TM: The dynmics of glomerulr ultrfiltrtion in the rt. J Clin Invest 5: , ARENDSFIORST WJ, GOTTSCHALK CW: Glomerulr ultrfiltrtion dynmics: Euvolemic nd plsm volume-expnded rts. 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