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Kidney International, Vol. 6 (1974), p. 24—37

Medullary plasma flow and intravascular leukocyte accumulation in acute renal failure

KIM SOLEZ, ELIZABETH C. KRAMER, JENNIFER A. Fox and ROBERT H. HEPTINSTALL

Department of Pathology, The Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland

Medullary plasma flow and intravascular leukocyte accumula- It is well established that renal cortical blood flow is tion in acute renal failure. The accumulation of nucleated cells in markedly reduced in acute renal failure [1—5] but the the in acute renal failure has been attributed to intra- vascular hematopoiesis caused by reduced medullary blood situation with regard to medullary blood flow is less flow. Medullary plasma flow was determined in rats with HgCI2- clear. Inert gas washout techniques for measuring induced acute renal failure by infusing '251-albumin iv. for 15 renal blood flow have demonstrated an apparent or 30 sec and measuring radioactivity in plasma and the renal papillae. Measurements made 40 to 48 hr after i.v. injection of increase in medullary blood flow in acute renal failure HgCI2 revealed a significantly raised medullary plasma flow at a [1, 4, 5] but this has been attributed to a merging of the time when there were prominent collections of nucleated cells in the vasa recta. Vascular tracer studies using India ink demon- cortical and medullary components of the washout strated that blood flow was maintained in vasa recta which curves rather than to a true increase in medullary contained nucleated cells. Electron microscopic and histo- blood flow [1, 5]. chemical studies showed that these cells were lymphocytes, neutrophils and monocytes, present in approximately the same One very common morphologic feature of acute proportions as in the peripheral blood. It is suggested that the renal failure which has been interpreted as suggesting nucleated cells in the vasa recta are not produced within the that medullary blood flow is decreased is the presence , but are brought there via the circulating blood. Their accumulation within the vasa recta is unexplained but is un- of large numbers of lymphocytes, neutrophils and related to a reduction in medullary blood flow. other nucleated cells in the vasa recta of the . In an autopsy series, this lesion was observed Debit médullaire et accumulation intravasculaire de leucocytes dana l'insuffisance rénale aiguë. L'accumulation de cellules in 63 of 66 unselected cases of acute renal failure of nucldées dans les vasa recta au cours de l'insuffisance renal aiguë more than one day's duration and was sometimes the a etc attribuée a l'hématopoIEse intravasculaire déterminde par only definite histologic lesion present [6], a fact noted le diminution du debit sanguin médullaire. Le debit médullaire a ete dCterminé chez des rats atteints d'insuffisance renale aigud previously by Finckh, Jeremy and Whyte [7]. Baker induite par HgC12 au moyen d'une perfusion intraveineuse [8] attributed the accumulation of nucleated cells in the d'albumine 1251 durant 15 ou 30 seconds et de la mesure de Ia vasa recta to intravascular hematopioesis. Other radioactivité du plasma et de la papille rénale. Les détermina- tions faites 40 a 48 heures aprés l'injection intraveineuse de investigators [9—12] postulated that the nucleated cells HgC12 montrent une augmentation significative du debit were released into the circulating blood by the bone medullaire contemporainc de l'existence de quantitds impor- marrow, spleen and lymph nodes and then became tantes de cellules nucléées dans les vasa recta. Les etudes vascu- laires a l'aide de l'encre de chine comme traceur dCmontrent que lodged in the vasa recta due to hemodynamic factors. le debit sanguin est maintenu dans les vasa recta qui contiennent Both Baker [8] and Remmele [12] considered this des cellules nucleCes. La microscopie électronique et les etudes lesion to be the result of decreased medullary blood histochimiques montrent que ces cellules sont des lymphocytes, des neutrophiles et des monocytes, approximativement dans les flow. mêmes proportions que dans le sang périphérique. Ii est suggéré Recent review articles discussing the circulation of que les cellules nucléées des vasa recta ne sont pas produites dans le rein mais y sont apportées par Ia circulation sanguine. Leur the kidney [13—15] have emphasized the technical accumulation a l'intérieur des vasa recta n'est pas expliquée mais and interpretive difficulties inherent in the measure- elle n'est pas Ia consequence d'une diminution du debit sanguin ment of medullary blood flow using methods currently medullaire. available. In an effort to overcome these difficulties, we have developed a simple method of determining Received for publication December 4, 1973; and in revised form February 4, 1974. medullary plasma flow in the rat based on the radio- © 1974, by the International Society of Nephrology. active albumin infusion methods of Lilienfield, Mag-

24 Medullary plasma flow in acute renal failure 25 anzini and Bauer [16] and Ganguli and Tobian [17]. remained above 90 mm Hg were accepted. A midline This paper describes the use of this method in the abdominal incision was made and loose, nonocclusive investigation of renal medullary hemodynamics in 1—0 silk ties were placed around the renal pedicles. acute renal failure induced by mercuric chloride, and Using a syringe pump (Sage Instruments, Cambridge, the relationship of medullary plasma flow to the MA) a solution of '251-albumin (Squibb and Sons, accumulation of nucleated cells in the vasa recta. In Princeton, NJ) (1 tCi/ml) in 0.9% NaCI, to which a addition, medullary plasma flow was measured after small amount of sodium fluorescein had been added, saline loading and in shock. was infused into thejugular vein at a rate of0.37 ml/min under UV light until 30 sec after the appearance of yellow fluorescence at the kidney hilus. Jugular vein-to- Methods kidney circulation time proved to be 2.5 to 3.0 sec in Female Holtzman rats weighing 180 to 220 g were all experimental groups except for the shock animals, housed in pairs, or singly in metabolism cages, and in which it averaged 4.0 to 5.0 sec. At the end of the fed a routine diet of standard laboratory chow infusion period, the ties around the renal pedicles (Purina, Ralston Purina Co., St. Louis, Mo) and un- were pulled tight. A continuous sample of arterial limited tap water. blood (total volume, 0.3 to 1.0 ml) was collected at a Determination of medullary plasma flow.1 In prelim- constant rate through the carotid cannula,, for the final inary experiments using the method of Lilienfield et al 30 sec of the infusion period. This procedure resulted [16] as modified by Ganguli and Tobian [17] for the in the radioactive albumin circulating through the rat, we observed a great deal of apparent variation in kidneys for a 30-sec period which coincided with the medullary blood flow among control animals, con- blood collection period. For the HgCl2-treated group, firming the experience of the original investigators who in which papillary transit time was less than 30 sec, a described these methods. We were able to reduce this 15-sec period was used as well. variability by infusing the radioactive albumin intra- The kidneys were removed with the pedicle ties venously instead of intraarterially. Further modifica- intact and cut into slightly uneven "halves" from pole tion of the necessary surgical techniques resulted in to pole so that all of the papilla remained in just one a method which gave reproducible results. half. In the kidneys of all experimental groups, the The method is based on the fact that the transit time papilla could be readily distinguished from the outer for circulating albumin in the renal papilla of the medulla. The papillae were cut out using small normal rat is about 40 sec (see Results section). If dissecting scissors, wiped lightly against nonabsor- radioactive albumin is allowed to circulate through bent paper to remove adhering blood and placed into the kidney for 30 sec, the accumulation rate of capped, preweighed scintillation counting tubes.2 papillary radioactivity during this period is propor- The remaining portions of the kidneys were fixed in tional to plasma flow rate. This determination of buffered 10% formaldehyde solution and prepared for medullary plasma flow does not take into account histologic examination, which confirmed that the glomerular filtration. However, since the glomerular papilla had been removed and the outer medulla left filtrate is largely reabsorbed before the papillary intact. The hemocrit value of the blood sample was portion of the descending is reached, it determined and an aliquot of the blood sample was contributes directly to medullary plasma flow. If placed in a scintillation counting tube. After weighing glomerular filtration is not taken into account, there is the papillae, the radioactivity of the blood sample and less than a error in the determination of medullary of the papillae was measured in a gamma well plasma flow (see Discussion section). scintillation counter (Baird-Atomic Inc., Cambridge, To determine medullary plasma flow, the rats were MA) and expressed as cpm/ml of plasma and cpm/ anesthetized with sodium pentobarbital (50 mg/kg) 100 g of papilla, respectively. (Sample height did not i.p. and placed on a warmed platform to maintain exceed 1 cm, thus minimizing geometric error in normal body temperature (37.5 to 38.8°C). The trachea, gamma counting [18].) By dividing papillary radio- left jugular vein and right carotid artery were cannu- activity by plasma radioactivity, medullary plasma lated. Carotid artery pressure was monitored using a pressure transducer (Sanborn Co., Waltham, MA). Except for the group of animals with hemorrhagic 2Ina departure from the method of Lilienfield et al [16], we did not quick-freeze the kidneys before sectioning them, since shock, only experiments in which mean blood pressure preliminary experiments with control animals produced repro- ducible results whether the kidneys were used in the fresh or in 1Themethod described measures inner medullary (papillary) the frozen state (31.34± 8.34 (SD) mI/min/lOO g for 12 frozen plasma flow, referred to in this paper as "medullary plasma kidneys vs. 32.22±5.16 (SD) mI/mm/bOg for 22 unfrozen flow". kidneys). 26 Solez et a! flow was determined, expressed in units of ml plasma/ via the carotid cannula to produce mean blood min/100 g of papilla. pressures of 30 to 70 mm Hg after a 30-mm equilibra- Determination of papillary transit time for albumin. tion period. Medullary plasma flow was then deter- To determine the approximate transit time for circu- mined as already described. lating albumin in the renal medulla, 20 normal rats and Saline loading. Each of eight normal rats received 27 rats with HgCI2-induced acute renal failure were an i.v. infusion of 0.9% NaCI equal to l0% of its prepared as already described for medullary blood body wt at a constant infusion rate over a period of flow determination. After '251-albumin had been one hour, after which medullary plasma flow was allowed to circulate through the kidney for 15,30,45 or determined. 60 sec, the was interrupted and the Vascular tracer studies. In 13 rats, acute renal failure apparent volume of distribution of albumin in the renal was induced with mercuric chloride (5 mg/kg) as papilla was determined by comparing papillary radio- already described. Forty-eight hours after mercuric activity to average plasma radioactivity. The time at chloride injection, a midline abdominal incision was which volume of distribution leveled off was taken to made and loose ties were placed around the renal represent transit time for albumin. pedicles. One milliliter of India ink diluted 1: 1 with Induction of acute renal failure. With the animal 0.9% NaC1 was injected into the inferior vena cava. under ether anesthesia, after a small lateral neck The ties around the renal pedicles were pulled tight 5, incision had been made, mercuric chloride solution 10, 15, 20, 25, 30 or 35 sec after ink injection (N=4 for (2 mg/mI of saline solution) was injected into the 15- and 20-sec groups; N= 1 for other groups). Both jugular vein of rats in a dose of 5 mg/kg. The incision kidneys of each rat were removed and prepared for was closed with wound clips and the animals placed in histologic examination. An indentical procedure was metabolism cages for urine collection under mineral carried out in seven normal rats. oil. Urine albumin concentration was determined White blood cell and differential counts, histochemical using Albustix (Ames Co., Elkhart, IN) and urine studies. In five control rats and seven rats with renal output for the 24-hr period preceding determination of failure 42 to 48 hr after the injection of mercuric medullary plasma blood flow was measured. The same chloride (5 mg/kg), white blood counts were deter- determinations were also carried out on six control mined in duplicate on heparinized blood samples rats. Medullary plasma flow was determined at 40 to using a hemocytometer (American Optical Co., 48 hr after mercury injection, a time at which there Buffalo, NY). Differential counts were done in tripli- were numerous nucleated cells in the vasa recta. Serum cate (300 cells counted for each animal) using smears urea nitrogen and serum creatinine concentrations prepared with Wright's stain. Smears of peripheral were determined on blood obtained at the time of this blood and 4 tfrozensections of the kidneys from six procedure using an autoanalyser (Technicon Corp., rats with acute renal failure 48 hr after HgCI2 treat- Tarrytown, NY) as previously described [19]. ment were stained with Giemsa for differential counts. Test for glomerular or tubular albumin leak. To In an additional similar group of six rats with acute renal determine whether substantial amounts of radioactive failure, blood smears and kidney frozen sections were albumin might be entering the papilla via the glomeru- stained for acid phosphatase using a modification of the lar filtrate during the 30-sec infusion, the of six diazo method of Barka and Anderson [20]. This was rats injected with mercuric chloride, 5 mg/kg 42 to 48 done to distinguish between monocytes (acid phos- hr before, were catheterized with PE 10 tubing phatase positive) and other large mononuclear cells. In (Intramedic, Clay Adams, Parsippany, NJ). After i.v. each animal a differential count was performed on 300 injection of 0.18 Ci of 1251-albumin (the amount white blood cells in the kidney vasa recta and 300 used for medullary plasma flow determinations), white blood cells in the blood smear. urine flow rate and radioactivity of the urine samples Preparation of tissues for light and electron micro- obtained during the next four hours were determined. scopy. Paraffin-embedded kidney tissue which had Determination of papillary water content. The water been fixed in I 0% buffered formaldehyde solution was content of the papillae of eight control rats and eight cut at 4 tandstained with hematoxylin-eosin, periodic rats with acute renal failure 48 hr after HgCl injection acid-Schiff and Giemsa stains. Tissue taken for elec- was determined by comparing wet weight to dry weight tron microscopy from six rats with acute renal failure after drying the tissue to constant weight in an oven at and six control rats, was fixed in 3% glutaraldehyde in 80CC. 0.1 M cacodylate buffer, postfixed in 2% osmium Induction of hemorrhagic shock. Thirteen normal rats tetroxide and embedded in epoxy resin (Epon) using were prepared for medullary plasma flow determina- methods previously described for this laboratory [21]. tions and samples of 3 to 4 ml of blood were removed Sections were stained with lead citrate and uranyl Medullary plasma flow in acute renal failure 27 acetate and examined in an electron microscope 38 (AEI-801, AEI Scientific, Falls Church, VA). Statistical treatment. Student's t test was used to 34 evaluate the significance of differences between 30 / means in the experimental groups [22]. 26 //. — 22 Results / -— 18 // V Albumin transit time. Studies of the apparent volume / 14 of distribution of infused 1251-albumin in the renal 7— //: papillae of 20 normal rats (40 kidneys) over time 10 // // showed a linear increase which leveled off about 40 6 / sec after the beginning of the circulation of radio- / .7/ active albumin through the kidney (Fig. 1). This sug- 2/ gested that the normal papillary transit time for 0 15 30 45 60 albumin is 40 sec, a result consistent with the earlier Time, see work of Kramer, Thurau and Deetjen [23, 24] demon- Fig. 2. Volume of distribution of radioactive albumin in the papillae of 24 rats with HgCl2-induced acute renal failure at strating an Evans blue transit time of 45 sec in the different times after the infused '251-albumin first entered the papillae of the dog. In the HgC12-treated group, renal arteries. Apparent circulation time is more rapid than in transit time was less certain since volume of distribution control rats and rate of accumulation of labeled albumin is did not increase in a linear fashion (Fig. 2), possibly greater, whether determined at 15 or 30 sec. because of increased intrarenal heterogeneity of blood flow. Using the intersection of the slope of the infusion time, was found to average 32.22± 1.10 initial rise with slope of the plateau at 45 to 60 sec as (sEM) ml/min/100 g of papilla in 11 control rats (Fig. 3), the transit time, a transit time of 24 sec was obtained a value comparable to that reported by Lilienfield et a! for the HgCl2-injected group. (A transit time of 30 sec [16] (25.44 ml/min/lOO g) and by Ganguli and Tobian is obtained if the average slope during the first 30 sec [17] (38.0 ml/min/100 g). The average absolute is used instead of the slope of the initial rise.) Because differences between the values for the two kidneys of a of this short transit time, the results of the 15-sec single animal was 4.70 1.42 (sEM) ml/min/100 g. infusions were used to determine medullary plasma flow in this group. The maximum volume of distribu- 64 tion was approximately the same in the HgC12-treated 60 group as in the control group. 56 Medullary plasma flow. Medullary plasma flow, 52 determined as already described using a 30-sec ,-., 44 26 40 36 22 0 28 18 -BP J50—70mm Hg

14 —— I // BP30-49 mm Hg

/ Control Shock Saline HgCI2 15 30 45 60 loading (40—48 hr) Time, see Fig. 3. Medullary plasma flow in experimental and control groups Fig. 1. Volume of distribution of radioactive albumin in the determined by using a 30-sec infusion of radioactive albumin. papillae of 20 control rats at 15, 30, 45 and 60 sec after the Lines connect the values for the left and right kidneys of indivi- infused '251-albumin first entered the renal arteries. The apparent dual animals. Medullary plasma flow is significantly elevated in circulation time for albumin is 40 sec. Volume of distribution the rats with HgCI2-induced acute renal failure even though increases linearly for the first 30 sec, suggesting that little medullary plasma flow is probably underestimated using the radioactive albumin leaves the papilla during this time. 30-sec infusion period in this group (see Fig. 4). 28 Solez et a!

Medullary plasma flow was significantly elevated 0.0008 [SEMI g for controls), they had an even more ele- (P<0.00l) in eight saline-loaded rats (53.16± 1.94 vated total papillary plasma flow (11.02 1.20 l.tl/min/ [SEMIml/min/100g), and significantly reduced (P< papilla) compared to controls (5.57±0.19 [sEM] 1/ 0.001) in eight rats with severe hemorrhagic hypo- min/papilla). Water content of the papillae of control tension and mean blood pressures of 30 to 49 mm Hg rats was 76.8± 1.4 (sEM)% as compared to 85.4±0.8 (11.16± 1.56 [sEM] mi/mm/bOg). Five rats with less (sEM)% in rats with acute renal failure, confirming that severe hypotension (mean blood pressure, 50 to 70 the weight difference was due to edema. mm Hg) had nearly normal medullary plasma flow At the time that medullary plasma flow was deter- (29.00 1.00 [SEMI ml/min/l00 g). In seven rats with mined in the mercuric chloride-treated rats, the acute renal failure 40 to 48 hr after mercuric chloride animals were virtually anuric and had serum urea injection, meduliary plasma flow, determined using a nitrogen concentrations ranging from 276 to 312 mg/ 15-sec infusion time (Fig. 4), was significantly elevated 100 ml (mean, 298 10 vs. 13.7 2.2 for controls) and (P< 0.001) expressed in terms of papillary weight serum creatinine concentrations ranging from 6.2 to (58.35 6.08 [SEMI ml/min/100 g. (Using a 30-sec in- 7.4mg/lOOml (mean, 6.80±0.16 vs. 0.60±0.07 for fusion period [Fig. 3], a meduilary plasma flow in ten controls). Stools were black, semi-liquid and guaiac rats with renal failure was underestimated as 40.50 positive. Hematocrit values averaged 36.41.2 (sEM)% 1.06 [sEM] ml/min/lOO g, a result still significantly as compared with control values of 46.7 0.7 (sEM)%. different from the control value [P<0.01J.) Since The rats were lethargic and anorexic, but had normal papillary weight was increased approximately 9% blood pressures. in these rats (0.01888 0.0010 [SEMI g vs. 0.01728 No evidence of increased glomerular leakiness to albumin in rats with mercuric chloride-induced acute 100 renal failure was found. Urine samples collected in the 96 24-hr period before medullary blood flow determina- 92 tion contained 2 to 3 albumin (100 to 300 mg/ 88 100 ml) as compared with trace to 1 +albuminin con- 84 trols (15 to 30 mg/100 ml). However, urine output was 80 so markedly reduced in the rats with acute renal 76 failure (1.02±0.35 [SEMI ml/24 hr vs. 11.33± 1.87 / [sEM] ml/24 hr for controls) that total albumin 72 excretion was not increased. In six rats which received 68 an i.v. injection of '251-albumin 48 hr after acute renal 64 failure was induced with mercuric chloride (5 mg/kg), :: 60 the urinary excretion rate of radioactivity was so low E56 (0.3 to 0.8 cpm/30 sec) that it could not have accounted 52 for more than a 0.25 increase in apparent medullary o 48 / blood flow. It therefore seems unlikely that radio- 44 active albumin brought to the papilla via the glomeru- 40 lar filtrate constituted an important source of error in 36 the determination of medullary plasma flow in rats with nephrotoxic acute renal failure. 0 32 28 Vascular tracer studies. In animals with acute renal 24 failure 48 hr after the injection of mercuric chloride, 20 India ink was found in vessels of the and outer medulla five seconds after injection into the 16 inferior vena cava. At ten sec it could be seen in a few 12 of the arterial vasa recta of the papilla. By 15 sec most 8 of the arterial vasa recta of the papilla contained 4 India ink and this material could be seen in the venous 0 vasa recta of the outer medulla which contained large Control HgCI2 numbers of nucleated cells. At 20 sec, India ink ap- (40—48 hr) peared for the first time in the venous vasa recta of the Fig.4. Medullaryplasma flow in rats with HgCI2-induced acute papilla which contained many nucleated cells (Fig. 5). renal failure, determined by using a 15-sec infusion of radioactive albumin. Data from control rats using a 30-sec infusion is given By 30 sec after injection, the vasa recta were diffusely for comparison. filled by india ink. Munkacsi [32]. criteria ofMoffat[30],Leverand Kriz[31],andNewstead mercuric chloride-induced acute renalfailurethanin medulla appearedtobe more rapidinratswith studies ofBaker[8].TheIndiainkflowrateinthe vasa recta,afindingconsistentwiththeserialsection recta,3 andrelativelyscarceinthearterial(descending) cells wereabundantinthevenous(ascending)vasa accumulations ofnucleatedcells.2)The flow throughthesamevasarectawhichcontained tracer studies:1)Therewasmaintenanceofblood 30 sec. of thepapillaat20secandinvenousvasarecta with Indiainkfirstappearinginthearterialvasarecta but apparentcirculationtimewassomewhatslower, eosin, )< can beseen(arrows),AttheupperrightisavasrectumcontainingleukocyteswhichhasnotyetfilledwithIndiaink(hematoxylin- Fig. 5.VasarectainthepapillaofaHgCl2-treatedrat,20secafteri.v.injectionIndiaink.Leukocytesintermingledwith ink casts 44hrafterHgCl2injection(Giemsa,x370). Fig. 6.Numerousleukoc.vtes(arrowheads)inthevasarectaofoutermedullaadjacenttodamagedtubulescontainingnumerousdense Venous andarterialvasarectawere distinguishedusingthe Two conclusionscouldbedrawnfromthevascular In controlanimals,thesamesequencewasobserved 650). Medullary plasmaflowinacuterenalfailure of theleukocytes intheperipheralblood and5.5%of all celltypesexceptmonocytes, whichmadeupI.9% recta wasverysimilartothat intheperipheralbloodfor demonstrated thattheleukocyte populationinthevasa frozen sectionsfromsixrats withacuterenalfailure study ofGiemsa-stainedblood smearsandkidney lymphocytes andmonocyteswasalsoobserved.A renal failure.Aslightincreaseinthenumbersof leukocytes incontrolanimalsand40%ratswith neutrophils whichconstitutedl0%oftheblood primarily toaseven-foldincreaseinthenumberof As demonstratedinTable1,thisleukocytosiswasdue 298 cells/mm3vs.9,435669forcontrols). mercuric chloridewassignificantlyelevated(18,192 studies. Theaveragewhitebloodcountinratswith acute renalfailure40to48hraftertreatmentwith be accuratelymeasuredwiththistechnique. controls, butitisrecognizedthattransittimecannot White bloodcountanddifferential,histochemical

- .'1' :.t 29 30 Solez et al

Table 1. Total white blood cell and differential counts in control rats and rats with acute renal failurea

Total Neutrophils Monocytes Lymphocytes Eosinophils leukocytes Acute renal failure 18,192± 7,313± 491 10,206± 182± 298/mm3 373/mm3 61/mm3 320/mm3 36/mm3 (40.2±2.0%)(2.7±0.3%) (56.1 1.8%) (1.0±0.2%) Control 9,435±669/mm3997±66/mm3283±85/mm37,982± 179/mm3174±47/mm3 (10.6 0.7%)(3.0 0.9%) (84.6I .9%) (1.8 0.5%)

Values indicate 15EM.

Table 2. Differential white blood cell counts on peripheral and vasa recta blood in rats with acute renal failure

Neutrophils Monocytes Lymphocytes Eosinophils Peripheral 34.0±2.3% 1.9 0.2% 63.8 2.1% 0.2±0.1% Vasa recta 33.4±2.0% 5.5±0.5% 60.4±2.1% 0.3±0.1%

a Differentgroups of animals were used to derive the data in Tables 1 and 2. b Values indicate I SEM. the leukocytes in the vasa recta (Table 2). Acid distinguished from the descending vasa recta by their phosphatase stains of blood smears and kidney frozen larger size and the presence of endothelial fenestrae3 sections from six additional rats with HgCI2-induced (Fig. 8). Leukocyte-to-leukocyte contacts were fre- acute renal failure confirmed this difference in the pro- quently observed. In many cases thin cytoplasmic portion of monocytes. In the vasa recta, 6.4± 1.4 processes were seen extending from a leukocyte to- (sEM)% of the leukocytes were acid phosphatase ward an endothelial cell in a way that suggested that positive cells morphologically consistent with mono- the surface coats of the two cells were in contact. In cytes; in the peripheral blood only 2.3 0.5 (sEM)% of contrast to this very loose contact between leukocytes the leukocytes were of this type. In both the vasa and the endothelium of the ascending vasa recta, recta and the peripheral blood, a small proportion of several of the few leukocytes that were observed in the the leukocytes (1.0±0.3 [SEM]% and 1.4±0.6 [sEM]%) descending vasa recta could be seen to be more firmly weremorphologically consistent with monocytes but attached to the wall of the vessel and surrounded by acid phosphatase negative. Nucleated red blood cells platelets in areas of apparent endothelial discontinuity made up less than 0.2% of the nucleated cells in the (Fig. 9). Leukocyte accumulations were observed in peripheral blood of animals with acute renal failure and the ascending vasa recta of both the outer and the were not observed in the vasa recta. inner medulla. No cells were seen which were morpho- Histology. In pilot studies, the first unequivocal logically consistent with normoblasts or white blood accumulation of nucleated cells in the vasa recta, with cell precursors, and no mitoses were observed. No the number of leukocytes exceeding the number of red changes were observed in endothelial cells which might cells in some vessels, was found at 36 hr after HgC12 have suggested transformation into leukocytes. injection. By 40 to 48 hr, the time of which the studies The other light microscopic renal morphologic described in this paper were carried out, the lesion was changes were similar to those described in mercury- much more prominent (Fig. 6). induced acute renal failure by other authors [25—29]. Electron microscopic findings confirmed that the There were obvious necroses of the mid and terminal leukocytes in the vasa recta were of three types (Fig. portions of the proximal tubules with numerous dense 7): neutrophils, lymphocytes and monocytes. The eosinophilic casts. Many tubules appeared dilated, and segmentation of the neutrophils was variable. Some there was extensive interstitial edema. Collections of had two rather coarse lobes, while most had multiple leukocytes were frequently found in the venous vasa lobes. Leukocyte accumulation occurred predomin- recta adjacent to necrotic tubules. However, no inter- antly in the ascending vasa recta, which could be stitial inflammatory infiltrates were seen. Medullary plasma flow in acute renal failure 31

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Fig.7. Monocytes (M), lymphocytes (L) and neutrophils (N) in an ascending vas rectum of a rat 46 hr after HgC12 injection. The leuko- cytesappear to be making loose contact with the endothelium and with each other (lead citrate and uranyl acetate, x 7,000). 32 acetate, x17,700). There isnoflatteningoftheneutrophils againstthevesselwallandendotheliumappearsintact throughout(leadcitrateanduranyl Cytoplasmic projectionsoftheleukocytes extendtowardtheendothelium(arrows)andmakecontact withotherleukocytes(arrowhead). Fig. 8.4neutrophil(N)andamonocyte (M)inanascendingvasrectumwithcharacteristicfenestrae (F)46hrafterHgCI2injection. - a. -. t ______A I'/ I Solez etal

4, if II

L1 34 Medullary plasma flow in acute renal failure 33

groups are easily demonstrated. The standard devia- tion of the mean in our control group (5.16 ml/min/ 100 g) is considerably less than that of Ganguli and Tobian (11.40 ml/min/l00 g) [17] and Lilienfield et al (8.02 ml/min/l00 g) [16]. There is excellent agreement between values for the right and left kidney in the same animal. Third, since the method involves weighing the papilla, alterations in tissue volume can be corrected for and do not represent an uncontrolled variable as is the case in several other methods [13, 40]. Both total plasma flow and plasma flow per unit weight may be calculated. Fourth, small differences among animals in the concentration of radioactive indicator in arterial blood are automatically corrected for by using a carotid arterial blood sample from which one may accurately estimate the average concentration of radioactive indicator in blood perfusing the kidney during the infusion period. Miscalculation of jugular vein to 'S circulation time could therefore introduce little error into the final calculation of medullary plasma flow. The use of an intravenous infusion assures good mixing of radioactive indicator with plasma, which is often inadequate with intraarterial Fig. 9. A lymphocyte (L) with accompanying platelets (P) attached infusions [41]. to the wall of a descending vas rectum 40 hr after HgC12 injection. The method used does not reflect differences in Note the characteristic fenestrae of the adjoining ascending vas rectum (F) (lead citrate and uranyl acetate, x 11,700). medullary plasma flow brought about by differences in the amount of plasma water filtered by the . Rather, it assumes that the concentration of albumin Discussion in plasma entering the papilla is the same as that in It is difficult to find a reliable means of determining systemic arterial blood. In the normal animal, renal medullary plasma flow [13—15, 33, 34] and it is filtration fraction is about 0.33 [42]. All but approxi- therefore necessary to examine critically the way in mately 1 5% of the glomerular filtrate is reabsorbed which this was done in the present study. The method before the papillary portion of the 1oop of Hnle is used is a simple one and several lines of evidence sug- reached [43] and, thus, contributes to medullary plasma gest that the results generated by this method are flow. Assuming that filtration fraction in juxta- valid. medullary is the same as that for the kidney First, the values obtained for medullary plasma flow as a whole, calculated medullary plasma flow would be in saline-loaded animals, in hemorrhagic shock and in approximately 5% greater than actual medullary control animals using this method are consistent with plasma flow in the normal rat. There is evidence that in the data obtained by other investigators using differ- hemorrhagic hypotension [44] and acute renal failure ent methods [13—17, 23, 24, 35, 36]. Medullary blood [45, 46] filtration fraction is markedly reduced, and it flow seems to be well maintained in moderate hypo- is likely that calculated medullary plasma flow tension (mean blood pressure, 50 to 70 mm Hg) con- approaches actual medullary plasma flow in these firming that medullary blood flow is autoregulated, as situations. This presumably would also be the case in suggested by Carriere et al [37] and Ganguli and saline-loaded rats, in which filtration fraction in deep Tobian [17]. In more severe hypotension (mean blood nephrons has been reported to be massively decreased pressure, 30 to 49 mm Hg) medullary blood flow is [47]. markedly reduced, in keeping with the findings of In the normal animal there is extensive leakage of Kramer [38] and Aukland and Wolgast [39]. albumin into the interstitial space where it equilibrates Second, within each of the experimental groups, with the large extravascular albumin pool [48—50]. The there is a relatively narrow range of values for medul- fenestrated ascending vasa recta are quite permeable lary plasma flow, so that the differences between to albumin and other small molecular weight proteins. 34 So!ez et a!

Shimamura and Morrison [51] have recently demon- Baker [8] and Remmele [12] attributed this pheno- strated extensive leakage of ferritin and horseradish menon to decreased medullary blood flow with local peroxidase out of the descending vasa recta 30 sec hematopoiesis occurring during shock. The present after intravenous injection in normal rats. There is no study, and the earlier work of Jaenike and Schnee- evidence that the conduit vessels bringing blood to the berger [52] and Rosen et al [53] demonstrate that this medulla are similarly permeable to albumin. It lesion is characteristic of acute renal failure in the deserves to be emphasized that extensive leakage of absence of shock. The absence of red blood cell and albumin into the papillary interstitium from the white blood cell precursors, and the similarity of the ascending vasa recta interferes in no way with the leukocyte population in the vasa recta to that in the determination of medullary plasma flow described in peripheral blood, argue against local production of this paper. It makes no difference whether the radio- leukocytes in the vasa recta and strongly suggest that active albumin brought to the medulla via the vasa the nucleated cells in the vasa recta are brought there recta is intravascular or extravascular at the time via the circulating blood. It is unclear whether or not papillary radioactivity is measured. The fact that the the accumulation of nucleated cells in the vasa recta is maximum volume of distribution for radioactive related to the polymorphonuclear leukocytosis obser- albumin is similar in rats with acute renal failure and ved in acute renal failure [54]. in control rats (Figs. 1 and 2) suggests that leakage The accumulation of nucleated cells in the vasa of albumin out of the vasa recta is not significantly recta occurs in the presence of elevated medullary greater in acute renal failure than it is in the normal plasma flow, a seemingly paradoxical situation. animal. Findings of India ink vascular tracer studies confirm The method used to determine medullary plasma that blood flow is maintained in the vessels containing flow is based on two major assumptions: 1) that radio- large numbers of nucleated cells. The lesion persists active albumin reaches the papilla only via medullary despite increased blood flow, probably because the blood flow, and 2) that little radioactive albumin nucleated cells are loosely attached to the walls of the leaves the papilla during the infusion period. There is vasa recta and to each other. Recently, we have obser- no evidence that substantial amounts of radioactive ved that in rats with unilateral mercuric chloride- albumin are brought to the papilla via the glomerular induced acute renal failure (with one kidney protected filtrate or other routes and, thus, it seems likely that by ureteral ligation [55]), there are prominent collec- the first assumption is valid. The fact that the volume tions of nucleated cells in the vasa recta in the affected of distribution of radioactive albumin increases kidney but not in the protected kidney. This is true linearly for the first 30 sec in control animals (and, thus, despite the fact that ureteral obstruction is associated that medullary plasma flow determined at 15 sec is the with a profound reduction in medullary plasma flow same as that determined at 30 sec; see Fig. 1) suggests (K. Solez, to be published). Thus, it appears clear that that the second assumption is upheld in this group. If that this lesion is not caused by reduced medullary substantial amounts of radioactive albumin left the plasma flow. papilla during the infusion period, then the plot of the The accumulation of leukocytes in renal medullary volume of distribution during the first 30 sec would be vessels is a local phenomenon rather than a systemic expected to be nonlinear, with a decreasing slope. In one. We have not observed a similar phenomenon in the group of animals with acute renal failure, it seems the vessels of the lungs, liver or intestinal villi of unlikely that substantial amounts of radioactive animals with nephrotoxic acute renal failure. albumin left the papilla prior to the time at which The accumulation of leukocytes in the vasa recta in medullary plasma flow was determined (15 sec; see acute renal failure is similar to the margination of Fig. 2), but if this were the case, medullary plasma leukocytes which occurs as a part of the acute inflam- flow would be underestimated in this group. matory response. However, margination results in the The method used in the present study to measure preferential accumulation of neutrophils [56], while medullary plasma flow seems to be both more reliable in acute renal failure neutrophils and lymphocytes and subject to fewer interpretive difficulties than the accumulate in the vasa recta in the same relative pro- other methods which have been described in the portions as in the peripheral blood. Flattening of literature [13—17, 23, 24, 33]. It is likely that this leukocytes against the endothelial surface and sub- technique will be found useful in the study of medul- sequent diapedesis is not observed in the vasa recta in lary hemodynamics in other disease states besides acute renal failure. Since the rate of movement of acute renal failure. marginated leukocytes is proportional to blood flow The accumulation of nucleated cells in the vasa rate, and since only slowly moving leukocytes emi- recta is a conspicuous feature of acute renal failure. grate out of vessels [57], the elevated medullary blood Medullary plasma flow in acute renal failure 35 flow in acute renal failure may be one of the factors medullary plasma flow is increased in glycerol-induced which prevents leukocyte emigration from the vasa acute renal failure just as it is in the mercuric chloride recta. There is no evidence that the leukocytes in the model. If the increased medullary plasma flow demon- vasa recta are immobilized, and it seems likely that the strated in the present study of nephrotoxic acute renal leukocytes are in constant motion rolling along the failure also occurs in oliguric states associated with endothelial surface. less prominent renal structural damage, it may contri- It is possible that the accumulation of leukocytes in bute to the low urine osmolarity observed in these the vasa recta results from the accumulation in the states by washing out the medullary osmotic gradient renal medulla of a chemotactic substance in a situation [24]. Decreased outer cortical perfusion has come to in which the endothelial and hemodynamic changes be regarded as the "final common pathway" [70] of which favor firm sticking and emigration of leukocytes acute renal failure. Whether the increased medullary are not present. Preliminary results in this laboratory plasma flow demonstrated in this study will be found suggest that synthesis by the renal medulla of prosta- to be similarly universal in other types of acute renal glandin E (a potent chemotactic substance [58]) is failure remains an unanswered question pending increased in acute renal failure, a finding also reported further studies. by Torres, Strong, Romero and Wilson (personal communication). There is presently insufficient evi- dence to connect this finding with the phenomenon of Acknowledgments leukocyte accumulation in the vasa recta. It has been This work was presented in part at the 57th Annual suggested recently that transport of prostaglandins Meeting of the Federation of American Societies for from the renal medulla to the cortex via the venous Experimental Biology in Atlantic City, New Jersey, vasa recta may be an important factor in the autoregu- April 16, 1973. This study was supported by Public lation of renal blood flow [59]. Health Service grants HL-07835 and GM-00415. In 1947, Trueta et al [60] suggested that there was a Patricia Bulluck typed the manuscript and Marilyn shunting of blood away from the cortex through the Miller provided technical assistance. medulla in acute renal failure. The presence or absence of vascular pathways that would permit an aglomerular Reprint requests to Dr. Kim Solez, Department of Pathology, shunting of blood has been extensively debated [61— The Johns Hopkins Hospital, Baltimore, Maryland 21205, U.S.A. 63]. Vascular channels between the afferent and effer- ent arterioles bypassing the glomeruli have been de- References scribed by Ljungqvist [64] and Ljungqvist and Wager- 1. HOLLENBERO NK, EPSTEIN M, ROSEN SM, BASCH RR, OKEN mark [65] but their presence has recently been DE, MERRILL JP: Acute oliguric renal failure in man: denied by Spinelli et al [66]. Lameire, Schaper and Evidence for preferential renal cortical ischemia. 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