Journal of Clinical InvestigationVol. 44, No. 1, 1965

Prevention of Pyelonephritis by Water Diuresis: Evidencefor the Role of Medullary Hypertonicity in Promoting

Renal Infection *VINCENT T. ANDRIOLE t AND FRANKLIN H. EPSTEIN t

(From the Department of Internal Medicine, Yale University School of Medicine, NewHaven, and the Veterans Administration Hospital, West Haven, Conn.)

The medulla of the kidney is much more sus-ceptible to infection than the cortex (1-5). Theunique vulnerability of the renal medulla has beenascribed in the past to its anatomical location(6), to its relatively poor circulation (7), and toits chemical composition, in particular to its con-tent of ammonia and ammonia-producing en-zymes that inhibit the action of complement (8).The present experiments suggest that the sus-

ceptibility of the medulla to infection is deter-mined at least in part by another of its properties,namely, its habitual hypertonicity. A simplemethod for inducing chronic water diuresis inrats has provided a model for studying many as-pects of experimental pyelonephritis. Renal in-fection in rats can be prevented by decreasing thenormally hypertonic environment of the renalmedulla through sustained water diuresis.

Methods

Animals. White female Sprague-Dawley strain rats,weighing 150 to 200 g were used. Animals wereweighed on the first and last days of each experiment.All animals were fed Purina lab chow pellets and tapwater ad libitum.Water diuresis. Chronic water diuresis was induced

in rats by the simple method of adding 5%o glucose totheir drinking water. The volume of fluid intake and

* Submitted for publication July 14, 1964; acceptedSeptember 14, 1964.Presented in part at the joint national meeting of the

American Federation for Clinical Research and theAmerican Society for Clinical Investigation, AtlanticCity, N. J., May 1964, and at the Second InternationalSymposium on the Biology of Pyelonephritis, Boston,Mass., June 1964.Aided by grants E 1850, AI 05585, HE 00834, and

AM 06397 from the U. S. Public Health Service.t Clinical Investigator, Veterans Administration Hos-

pital, West Haven, Conn.t Recipient of a U. S. Public Health Service Research

Career Award.

urine output was measured daily for each rat. Urinesamples were analyzed for glucose with Clinitest re-agent and for osmolality with a Fiske osmometer.Samples of blood were obtained from the abdominalaorta of rats anesthetized with pentobarbital and wereanalyzed for glucose by the Nelson-Somogyi method(9). In some instances, urine samples were analyzed forpH with a Beckman pH meter, model G.

Organisms. The strains of Candida albicans (B-311)and Staphylococcus aureus (Giorgio) have been main-tained in this laboratory for 5 years, and details oftheir handling have been described previously (4, 10).The inocula were prepared in the following manner.Cells of Candida albicans from 2% glucose, 1% neo-peptone agar slants, incubated at 370 C for 24 hours,were suspended in physiologic saline. The number ofcells in this suspension was determined by direct hemo-cytometer counts in a Spencer Bright-Line Neubauercounting chamber, and serial dilutions in physiologicsaline were made so that the final inoculum contained106 organisms per ml, the exact number being deter-mined by standard pour plate enumeration techniques.The inoculum of Staphylococcus aureus was preparedfrom a nutrient broth culture, incubated at 37° C for18 to 20 hours, and contained 2 X 108 cells per ml.Each animal was challenged with a 0.5- or 1.0-ml inoculumof either Candida or Staphylococci, respectively, injectedinto a lateral tail vein. These inocula are known to in-fect the renal medulla of normal rats (11, 12).

Microbiological and pathological studies. Most ani-mals were killed 8 days after challenge. Some, how-ever, were killed at 2 and 6 hours and at 1, 2, and 5 daysafter intravenous Staphylococcal challenge.

Sterile technique was maintained during all surgicalprocedures. Under pentobarbital anesthesia, the ab-dominal wall was cleansed with 70% alcohol and in-cised so that the entire urinary tract was exposed.After urine and blood were collected in some animals,the kidneys, and in some instances, liver and spleen,were removed and placed in Petri dishes for macroscopicexamination and sectioning. A midline longitudinal sec-tion was made in some kidneys. One-half of the kidneywas fixed in 10% formol. These specimens were seriallysectioned, and every tenth section was mounted andstained with hematoxylin-eosin for histologic examina-tion. The remaining half of the kidney was placedin a Teflon tissue homogenizer and ground in 4.5 ml

73

VINCENT T. ANDRIOLE AND FRANKLIN H. EPSTEIN

of 0.85%o sodium chloride solution until a homogeneoussuspension was'obtained. Whole kidneys were culturedafter homogenization with 9.0 ml of saline. Theseconcentrations represented a 10' dilution. Subsequenttenfold dilutions were prepared in saline solution.Agar pour plates were made from these dilutions, andcolony counts were determined after incubation for 48hours.Each specimen of whole spleen or approximately 1 g

of liver was weighed, diluted 1: 10 in physiologic saline,homogenized, and quantitatively cultured as alreadydescribed.

Urine culture. Immediately after opening the ab-dominal cavity a small quantity of urine was aspiratedthrough the bladder wall. One loopful of this wasstreaked on blood and desoxycholate or trypticase soyagar. In certain animals serial dilutions of urine weremade in nutrient broth, and pour plates were made forfinal bacteriological counts.Blood cultures were taken by direct heart puncture.

Pour plates containing 0.1 ml of blood were incubatedfor 48 hours, and the number of colonies was recorded.

Criteria of infection. Kidneys were considered in-fected when they contained 5 X 104 or more colonies.Urine cultures were considered positive when ten ormore colonies were found on the streaked plate. Thisconcentration is equivalent to 103 to 10' colonies perml of urine. Blood cultures were considered positive

FLUIDINTAKEm/

URINEOUTPUTm/

8e

- CONTROLtollA I I LKIW. is

r-'%

r__Jo l l l

TABLE I

Prevention of Candida pyelonephritisby water diuresis

Infected/Candida Total* p

Control 16/17

Diuresis begun1 day before 3/17 <0.01inoculation

* The total number of rats inoculated intravenously with108 Candida albicans cells is shown, together with the num-ber of rats in each group that developed infection of therenal parenchyma.

when organisms were found in the blood agar pourplates.

Chemical studies of the renal medulla. The com-position of the renal medulla was determined in ratsduring water diuresis and compared to control animalsfed ad libitum. Rats undergoing water diuresis wereanesthetized with alcohol, and a suitable period wasallowed for inhibition of antidiuretic hormone by al-cohol before the kidneys were removed. The kidneyswere sectioned longitudinally and slightly off center sothat free access to the papilla and medulla was ob-tained. The entire white medullas of two kidneys(weight, 30 to 60 mg) were weighed, placed in 5 ml ofdistilled water, and homogenized with a motor-drivenTeflon pestle. Pieces of cortex weighing 250 to 400 mgwere homogenized in 10 ml of water. Immediately afterhomogenizing, samples were removed for duplicateanalyses of urea and ammonium (13). The homoge-nate was then heated at 1000 C for 1 hour, cooled, andcentrifuged, and analyses for sodium and potassiumwere done in duplicate on the supernatant fluid with aflame photometer. The water content of similar piecesof medulla and cortex was determined by weighing be-fore and after dessication at 950 C for 48 hours."Total solute" concentration in tissue was calculated

as 2 (Na++ K++ NH,+) + urea, where Na,+ K,+ NH4,+and urea are expressed as mmoles per kg tissue water.

CONTROL

>AX - x - x / Fxje)

I\

.\v WATER DIURESIS

I I--_._ /go i, I'rl

1 2 3 4 5 6 7 8 9 10DAYS

FIG. 1. MEAN DAILY LEVELS OF WVATER INTAKE,URINARY OUTPUT, AND URINARY OSMOLALITY (Uosm) IN 38CONTROL RATS FED AD LIBITUM AND 41 RATS GIVEN 5%0GLUCOSE TO DRINK. The arrow marked "challenge" in-dicates the time of Candida or Staphylococcal inoculation.

Results

Water diuresis produced by glucose ingestion.The mean daily levels of water intake, urinaryoutput, and urinary osmolality in 38 control ani-mals fed ad libitum and 41 rats given 5% glucoseto drink are shown in Figure 1. The first day'svalues are base-line observations. Rats continuallydrink large amounts of glucose water, apparentlybecause they like it. The glucose ingested is pre-sumably metabolized, since glucose does not ap-pear in the urine or in abnormal amounts in theblood. The average blood sugar in five ratsdrinking glucose for 2 days was 138 mg per 100

1400

U 1000

mOsm/kg 600

200

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41

PREVENTION OF PYELONEPHRITIS BY WATER DIURESIS

ml as compared to 142 mg per 100 ml in four ratsdrinking water. Water intake and urinary outputincreased several fold. Urinary osmolality droppedbelow 200 and frequently below 100 mOsm per kg.The pH of urine samples from control animalshad a mean value of 7.14 as compared to an aver-age of 7.88 for animals undergoing water diuresis.The weights of control rats at the beginning andend of the study period did not differ significantlyfrom those given glucose.

Candida pyelonephritis (Table I). An inocu-lum of Candida was injected into both groups atthe point marked "challenge" in Figure 1. Whenthe rats were killed 8 days later, pyelonephritiswas observed in 16 of 17 control animals and inonly three of 17 rats undergoing water diuresis.Gross abscesses, commonly seen in the kidneys ofcontrol animals, were absent in the kidneys of thethree infected rats undergoing water diuresis.

Staphylococcal pyelonephritis (Table II, Fig-ures 2 and 3). The incidence and severity ofStaphylococcal pyelonephritis were also dimin-ished by water diuresis. Pyelonephritis was ob-served in 20 of 21 control animals. Rats wereprotected from renal infection when water diu-resis was begun 1 day before or 1 day afterStaphylococcal inoculation, since only three of 12and four of 12 animals, respectively, were in-fected. When diuresis was begun as late as 3days after inoculation, infection was found inonly six of 13 rats killed 5 days later. Since re-nal parenchymal infection and abscess formationare known to occur regularly between 24 and 48

TABLE II

Effect of water diuresis in preventing Staphylococcal infectionof the kidneys

Infected/Staphylococcus* Total p

Control 20/2 1

Diuresis begun 1 day 3/12 <0.01before inoculation

Diuresis begun 1 day 4/12 <0.01after inoculation

Diuresis begun 3 days 6/13 <0.01after inoculation

Diuresis begun 5 days 6/8 0.50-0.30after inoculation

* 108 Staphylococcus aureus cells were injected intra-venously at the time of inoculation.

LOGSTAPHPERKIDNEY

7-

6-CONTROL

4 __________

3- WATER DIURESIS \

2 \\

w''.,I 2 3 4 5 6 7 82'6

FIG. 2. EFFECT OF WATER DIURESIS ON THE NUMBER

OF STAPHYLOCOCCI REMAINING IN THE KIDNEY AFTER IN-

TRAVENOUS INOCULATION. Each point represents the logof the median number of Staphylococci (ordinate) re-covered from 12 kidneys at intervals of hours and days(abscissa) after intravenous challenge.

hours after intravenous challenge with this strainof Staphylococcus (12), some rats in this groupmust have been cured of their infection. Al-though renal infection was not prevented or curedby water diuresis begun 5 days after intravenousStaphylococcal challenge, animals in this grouphad sustained a water diuresis for only 3 days,since they were killed 8 days after inoculation.Water diuresis may alter the normal distribu-

tion of blood flow to the kidney (14, 15). There-fore, it was essential to determine whether waterdiuresis altered the number of organisms initiallydeposited in the kidney. Groups of six controlsand six rats undergoing diuresis were killed atintervals after intravenous Staphylococcal chal-lenge. The results of quantitative cultures of kid-ney homogenates are plotted in Figure 2. Eachpoint represents the log of the median number oforganisms recovered from 12 kidneys in eachgroup. The horizontal line represents the log of50,000. Colony counts per kidney at or abovethis number, when observed 8 days after chal-lenge, have been shown to reflect renal parenchy-mal infection and abscess formation in previousstudies (11, 12).The number of organisms present in the kidneys

of controls and animals undergoing diuresis 2 and6 hours after inoculation was almost identical.At 1 day there was more than a log difference,which remained until the eighth day when viableStaphylococci were no longer detectable in kid-

75

VINCENT T. ANDRIOLE AND FRAN\KLIN H. EPSTEIN

} CONTROL

o__ o } WATER DIURESIS

DAYSFIG. 3. EFFECT OF WATER DIURESIS ON THE NUMBER OF STAPHYLOCOCCI

REMAINING IN THE LIVER AND SPLEEN AFTER INTRAVENOUS INOCULATION.

Each point represents the log of the mean number of Staphylococci (ordi-nate) recovered from the liver or spleen of six rats at intervals of hours

and days (abscissa) after intravenous challenge.

neys undergoing diuresis. Staphylococci didnot appear in the urine of control rats until 1day after inoculation, a time when renal paren-

chymal infection was grossly obvious. Staphylo-cocci never appeared in the urine of rats under-going water diuresis. These observations dem-onstrate that water diuresis does not merely washorganisms out of the kidney into the urine.

In contrast to the counts in kidney, colonycounts of viable Staphylococci in liver and spleenof control rats and animals undergoing diuresis

were similar when compared at these same inter-vals (Figure 3).To determine whether the renal medulla of

animals undergoing water diuresis elaborated a

bactericidal substance, extracts of renal medullafrom controls and animals underging diuresis were

separately mixed with ten Staphylococcal organ-

isms in sterile test tubes containing broth, placedon a rotator, and incubated at 370 C for 24 hours.The growth rate of Staphylococci was determinedby pour plate colony counts of serial 100-fold di-

TABLE III

Effect of water diuresis on the compositon of renal medulla and cortex*

Medulla Cortex Urine

Na K NH4 Urea Total Na K NH4 Urea Total Uosm Ureasolute solute

mEq/ mEq/ mEq/ mEqi mmoles! mmoles! mEq/ mEq/ mEql mEqi mmoles immoksi mOsm/ mmoles!kg H20 100 g kg kg kg H20 kg H20 kg H20 100 g kg H2O kg H20 kg H20 kg H20 kg H20 L

DS H20 H20 DSWater 123 83 60 23 62 476 61 22 92 35 20 389 219 16

diuresis12 rats 1-23 4:13 i6 A-6 4±40 ±92 ±5 ±1.5 ±6 ±5 46 439 498 ±10n=6

Control 215 120 61 30 489 1,101 68 23 100 27 16 408 2,004 1,23212 rats ±21 ±21 ±9 ±16 ±127 ±152 ±4 ±4 ±10 ±6 ±8 ±23 ±740 ±500n=6

p <0.01 <0.01 NS NS <0.01 <0.01 <0.01 NS <0.01 <0.01 <0.02 NS <0.01 <0.01

* Values are mean ± standard deviation.NS = not significant (p > 0.05); DS = dry solids; Uosm = urinary osmolality.

76

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PREVENTION OF PYELONEPHRITIS BY WATER DIURESIS

EFFECT OF WATER DIURESIS ONCOMPOSITION OF RENAL MEDULLA

_ CONTROL

m WATER DIURESIS

mM/kgTISSUEH20

[N lurea] [K+] [NH +] [ total osm."]2 (No*+ K+ NH4M)

+ urea

FIG. 4. EFFECT OF WATER DIURESIS ON THE COMPOSITION OF THE RENALMEDULLA. Medullary concentration of sodium, potassium, ammonia, urea,and total calculated solutes in control (normal) rats and rats undergoingwater diuresis.

lutions. Extracts of renal medulla from animalsundergoing diuresis did not inhibit bacterialgrowth.

Chemical composition of the renal medulla andcortex. (Table III and Figure 4). Water diu-resis was associated with a fall in the concentra-tion of sodium in inner medullary tissue from215 ± 21 to 123 + 23 mEq per kg of tissue water.The concentration of urea, which averaged 489 +

127 mmoles per kg of tissue water in control rats,declined to 62 + 40 mmoles per kg in rats under-going water diuresis. As in dogs during waterdiuresis (16), the content of sodium and urea per100 g of dry solids in medullary tissue also de-creased. Tissue concentrations of potassium andammonium were unchanged by water diuresis.Total calculated osmolality of medullary tissuewas reduced by water diuresis from 1,101 to 476mOsm per kg.

In contrast to medullary tissue, total soluteconcentration of the cortex was unaltered by wa-ter diuresis. Sodium and potassium concentra-tions in the water of cortical tissue fell slightly,whereas ammonium and urea levels rose.

Histology. Serial sections of kidneys fromcontrol rats examined histologically 24 and 48hours after Staphylococcal challenge showed nu-merous microabscesses in the cortex and medulla.Clumps of Staphylococci were observed in the

center of medullary abscesses. In contrast, themedulla of the kidneys of rats undergoing diure-sis was relatively normal at 24 and 48 hours, eventhough the cortex contained a few focal areas ofinflammatory cells.

Discussion

These studies in the rat demonstrate that Can-dida and Staphylococcal renal parenchymal infec-tion can be prevented, and suggest that Staphylo-coccal renal infection can be cured by decreasingthe normally hypertonic environment of the renalmedulla through sustained water diuresis. Thedata, together with much more accumulated previ-ously by others (16-21), indicate that the bloodand interstitial fluids of the medulla are hyper-tonic when the urine is concentrated, but approxi-mate isotonicity during water diuresis.The organisms used in the present study, al-

though not common causes of human pyelonephri-tis, were specifically chosen because they con-sistently produce acute pyelonephritis in the non-manipulated rat kidney. Although several typesof renal and extrarenal manipulations have beenshown to predispose the kidneys of experimentalanimals to pyelonephritis, these models were notused in the present study in an attempt to elimi-nate the possible effects of the predisposing in-jury on the chemical composition of the kidney,

177

VINCENT T. ANDRIOLE AND FRANKLIN H. EPSTEIN

and on the delivery and function of phagocytesand other serum factors that contribute to thenormal defenses of the renal medulla.The decreased incidence and severity of renal

infection observed in rats undergoing water diu-resis was clearly not due to an inadequate numberof organisms initially deposited in the kidney, nordid water diuresis merely wash organisms out ofthe kidney into the urine of these animals. Thedecreased susceptibility to infection of the kidneysof rats undergoing water diuresis did not ap-pear to result from a bactericidal substance in therenal medulla induced by diuresis, since extractsof renal medulla from animals undergoing diuresisdid not contain a substance bactericidal forStaphylococci.

There are a number of possible mechanisms bywhich hypertonicity might enhance the vulner-ability of the medulla to infection. OriginallyHamburger (22) noted that hypertonic salinesolutions inhibit phagocytosis. More recently,Chernew and Braude (23) observed that concen-trations of sodium and of urea, within the rangenormally found in the renal medulla, inhibit phago-cytosis of Escherichia coli by human leukocytes.Highly concentrated human urine similarly de-presses phagocytosis, whereas more dilute urinesdo not. Hypertonic saline has also been shown toinhibit the activity of complement (24). Perhapsboth phagocytosis and complement activity areenabled to proceed normally in the medulla of therat undergoing water diuresis, but are inhibitedin the medulla of the hydropenic animal.

Since water diuresis increases renal medullaryblood flow (14, 15), a third explanation may liewith the more rapid delivery, during water diu-resis, of greater numbers of phagocytes and otherserum factors that contribute to the natural de-fenses of the body. However, increased numbersof phagocytes were not observed in the medullawhen the kidneys of animals undergoing diuresiswere examined histologically at various intervalsafter Staphylococcal challenge.

Finally, these natural defenses, namely, anti-body, complement, and lysozyme, can injure thecell wall of most urinary pathogens so that theyform protoplasts (25-27). These protoplasts arelysed in a hypotonic or isotonic environment, butare preserved in hypertonic solutions (28). Pro-toplasts would presumably be lysed in the iso-

tonic environment of the medulla during waterdiuresis.

Although we have no direct evidence to sup-port these possible mechanisms, the results of ourstudies do support the view that the hypertonicenvironment of the renal medulla is important inthe development of pyelonephritis. They thusprovide an experimental rationale for the time-honored practice of "forcing fluids" in patientswith urinary infection. When hypertonicity isovercome by water diuresis, experimental renalinfections may be prevented or greatly ameliorated.

Summary

Chronic water diuresis was induced in rats byadding glucose to their drinking water. The con-centrations of sodium and urea and the total cal-culated osmolality of inner medulla were signifi-cantly reduced by water diuresis, whereas tissueconcentrations of ammonia and potassium wereunchanged.Water diuresis protected against two types of

experimental pyelonephritis, that produced byintravenous inoculation of Candida albicans and ofStaphylococcus aureus.

These data support the view that the habitualhypertonicity of the renal medulla is an importantdeterminant of the special susceptibility of thistissue to infection.

Acknowledgments

We gratefully acknowledge the technical assistanceof Ellen Cooper, Penny Howe, Nancy Sudvoy, andNadia Mykety.

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PREVENTION OF PYELONEPHRITIS BY WATER DIURESIS

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