Kidney International, Vol. 16 (1979), pp. 366—376

Isoelectric point of albumin: Effect on renalhandling of albumin

JAMES N. PURTELL, AMADEO J. PESCE, DAVID H. CLYNE, WILLIAM C. MILLER, andVICTOR E. POLLAK

with technical assistance of RUSSELL BRAMLAGE, THOMAS SNIDER, and PAUL VITALE

UniversityofCincinnati Medical Center and Veterans Administration Hospital, Division of Nephrology, Department ofInternal Medicine, Cincinnati, Ohio

Electrical charge is thought to be an importantfactor in the integrity of the glomerular barrier tofiltration of macromolecules [1, 2]. Studies thathave explored the integrity of the glomerular barrierto filtration have been done with heterologous trac-er or probe proteins and with polysaccharide mac-romolecules. Advantages to using the polysaccha-ride macromolecules are: they are readily available;they can be refined to give a defined molecular radi-us; most importantly, they are neither reabsorbednor secreted by the tubules. But they are poly-saccharides, and not proteins which are the natural-ly occurring macromolecules. In addition, as point-ed out by Rennke and Venkatachalam, molecularconformational changes may facilitate their transitacross the glomerulus [3]. Thus, the charge on thesepolysaccharide macromolecules can be controlled,but not the changes in their structure.

Protein macromolecules of various molecular siz-es have been used as tracers to study glomerulartransit [4—8], but these have had a wide range ofisoelectric points, and the importance of charge hasbeen appreciated fully only recently [7]. Althoughheterologous protein tracer studies have provideduseful information [8], we believe it desirable to usethe naturally occurring plasma proteins as tracermolecules.

In this study we examine albumin, the most abun-dant protein in the plasma, the protein playing themajor role in oncotic pressure maintenance, and

Received for publication March 12, 1979

0085-2538/79/0016—0366 $02.20© 1979 by the International Society of Nephrology

366

whose concentration in plasma is 40 mg/mi and inurine is 10 mg/mi or less [9—11].

We have shown previously that normal human al-bumin is handled by the rat kidney in a manner nodifferent from that of endogenous rat albumin [12].The present study was designed to examine the ef-fect of change in isoelectric point (p1) on renal han-dling of the albumin molecule. Normal human albu-min and two derivatives of human albumin withhigher isoelectric points were infused into rats un-der conditions in which the renal handling of thesetracer molecules could be examined, while the renalhandling of endogenous rat albumin served as con-trol.

Methods

Preparation of human albu,nin of increased isoe-lectric points. Albumin derivatization was carriedout by the method of Hoare and Koshland [13]. Thisreaction involved the activation of the carboxylgroups of human albumin by a water soluble carbo-diimide, l-ethyl-3-(3-dimethyl-amino-propryl)-car-bodiimide hydrochloride (EDC; Pierce, Rockford,Illinois), and the subsequent reaction of the activat-ed carboxyl groups with a nucleophile, ethylenediamine (EDA; Fisher Scientific Company, FairLawn, New Jersey). EDA solution was prepared byadding 66.8 ml of anhydrous EDA to 500 ml of dis-tilled water, cooling the solution to 25° C and ad-justing the solution to a pH of 4.75 with approxi-mately 350 ml of 6 N hydrochloric acid. Eight millili-ters of 25% human albumin (Cutter Laboratories,Inc., Berkeley, California) was dialyzed against dis-tilled water to remove preservatives and was addedto the EDA solution. The degree of EDA addition(cationization) to the protein was regulated by theamount of EDC added. To alter albumin so that the

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Effect of p1 on renal handling of albumin 367

p1 was 5.5 to 6.6, we added 0.565 g of EDC (0.0064M); to increase the p1 to 7.2 to 8.2, we used 0.725 g(0.0082 M). The reaction continued for 120 mm andwas then quenched by the addition of 30 ml of 4 Macetate buffer (pH, 4.75). The solution was dialyzedat 4° C against several changes of distilled water,lyophilized, redissolved in distilled water, dialyzedagain versus distilled water, and finally rely-ophilized and stored at 4° C.

Characterization of cationized albumin. To dem-onstrate that the altered species of albumin main-tained cross reactivity with albumin and to charac-terize them, we did the following procedures: (1)immunoelectrophoresis in agarose, with barbitalbuffer (pH, 8.7) in the Pfizer Poly E Film systemand with rabbit antihuman albumin antiserum (Beh-ring Diagnostics, Sommerville, New Jersey); (2)isoelectric focusing with the LKB 8100 column pre-pared according to the instruction manual [14](LKB Instruments, Inc., Rockville, Maryland) andthe Lauda K-2/RD coolant system (Brinkmann In-struments); (3) sodium dodecyl sulfate poly-acrylamide gel electrophoresis, according to themethod of Laemli [15], with an acrylamide elec-trophoresis cell (Savant Instruments, Inc., Hicks-yule, New York) and with unaltered human albuminused as the control; (4) Stokes radius (molecularsize) with a Sephadex G-l00 column calibrated withblue dextran, human albumin, ovalbumin, and cyto-chrome C.

Study of nephron transport of unaltered humanalbumin and cationized human albumin. Experi-ments were done in 14 female, Sprague-Dawley rats(each weighing 200 to 260 g) obtained from a singlesource. The rats were anesthetized intraperitoneal-ly with mactin® (120 mg/kg) and placed on a heatedlaboratory board; body temperature was main-tained at 37° C by an external heat source and mon-itored with a rectal thermistor and telethermometer.

The trachea was cannulated with a 10-mm lengthof polyethylene (PE-lOO) tubing. Both femoral andthe left jugular veins were cannulated with PE-lOtubing. Using Braun syringe pumps, we infused 3H-inulin into the right femoral, normal saline into theleft, and the experimental albumin solution into thejugular vein. We cannulated the left femoral arterywith PE-50 tubing to collect blood samples and tomonitor blood pressures with a Narco Biosystemphysiograph (desk model DMP-4B). The bladderwas exposed by laporatomy, and a funnel-shapedpiece of PE-60 tubing was tied securely into the ure-thral orifice. Urine specimens were collected viacatheter into preweighed urine vials; completeness

of collection was insured by gentle direct bladdercompression.

In each experiment, there was an initial 60-mmequilibration period during which normal saline wasinfused at the rate of 3.75 mL/hr (62.5 dJmin) tocompensate for the insensible losses [16]. 3H-inulinwas administered in a priming dose of 4.5 Ci per0.75 ml of normal saline per 1,5 mm, followed by acontinuous infusion of 3 Ci in 0.5 ml of normal sa-line per hour. At the end of the two 30-mm controlperiods, unaltered human albumin of p1 4.9 or al-tered human albumin of p1 5.5 to 6.6 and p1 7.2 to8.2 were infused into 5, 4, and 5 rats, respectively.Forty milligrams of each albumin was dissolved in Iml of normal saline and infused over 15 mm [12].There were four 30-mm experimental periods. Theinitial collection was started when the albumin in-fusion was begun. Blood samples were drawn at themidpoint of each collection period.

At the end of the experiment, the animals wereweighed, and a left nephrectomy was performed.Tissue was quick frozen on an aluminum flag in iso-pentane, precooled with liquid nitrogen, and storedat —70° C for immunofluorescent microscopy. Thesections were incubated with fluorescein conjugat-ed monospecific antisera to both human albuminand rat albumin, with appropriate blocking and ab-sorption controls. The findings were scored on asemiquantitative scale (0 to 4+).

Chemical analysis of specimens. The urine vol-ume collected during each of the six collection peri-ods was measured by weighing. Urine and serumsodium concentrations were measured in an In-strument Laboratories flame photometer. Os-molalities were measured on a Wescor 5100 vaporpressure osmometer (Logan, Utah). 3H-inulin wasmeasured with a Packard liquid scintillation spec-trometer.

Measurement of rat albumin and altered humanalbumins. Albumin concentrations in serum andurine were measured by the double antibody radio-immunoassay technique [17]. Because the alteredhuman albumins cross reacted with the antisera, on-ly the native human albumin was used as the radio-labeled tracer. Rat albumin (p1, 4.9) and human al-bumin (p1, 4.9) were radiolabeled by the chlora-mine-T method with a Sephadex G-25 column andsodium iodide 125J solution (Amersham, Arlington,Illinois) [10].

For use in diluting the materials, the following so-lutions were prepared: Phosphate-buffered saline(PBS), pH of 7.4, was prepared by dissolving 1.13 gof dibasic sodium phosphate, 0.258 g of monobasic

368 Purtell et a!

potassium phosphate, and 8.5 g of sodium chloridein 600 ml of water, with the final volume brought upto I liter with water. For antigen dilution, a PBS-ovalbumin buffer (pH, 7.4) was prepared by dis-solving 50 mg of ovalbumin (grade V, Sigma, St.Louis, Missouri) in 50 ml of PBS buffer. For anti-body dilution, a diluent of 1/10 normal rabbit serumin PBS-ovalbumin buffer (pH, 7.4) was prepared byadding 1 part of normal rabbit serum to 9 parts ofPBS-ovalbumin.

Antiserum to rat albumin was prepared as pre-viously described [10]. For use in the assay of hu-man albumin, rabbit anti-human albumin was ob-tained from Behring Diagnostics (Sommerville,New Jersey). Goat antirabbit globulin served as thesecond antibody, obtained through the courtesy ofDr. Laxmi Srivastava, Division of Endocrinology,University of Cincinnati Medical Center. The goatantiserum was diluted 1: 10 in the PBS.

To determine the optimal antibody dilution foreach assay, we arranged a standard curve with 125J..albumin diluted 1: 500 and standards of unlabeledantigen containing between 62 and 1000 ng for

500, 1: 1000, 1: 5000, 1: 10,000, 1: 20,000 di-lutions of antisera. The curve giving the most suit-able displacement was then chosen. The albuminderivatives did not readily displace the '251-labeledhuman albumin in the radioimmunoassay. A signifi-cantly greater amount of antigen was needed tocompete for the antibody. Thus, the assays for thealtered albumins were less sensitive, but were ade-quate for almost all serum and urine samples.

Standard curves were constructed by adding 100l (0.1 ml) of the 12I-albumin antigen, 100 pJ of ei-ther antirat albumin or antihuman albumin, and 100sl of the working solution containing either 10.0,7.5, 5.0, 2.5, 1.25 or 0.625 tg/ml. Disposable Cor-fling borosilicate culture tubes, 75 mm x 10 mm,were used. Each sample was measured in duplicate.

For assay of rat albumin in serum, the serum wasdiluted 1: 10,000 in ovalbumin-PBS. For assay ofhuman albumin and its derivatives in serum, theserum was diluted 1: 1000 in ovalbumin-PBS. De-termination of all albumins in urine was performedon dilutions of I : 100 or greater. One hundred mi-croliters of diluted serum or urine were handledas working solution.

All tubes were then incubated at 37° C for 1 hourand then incubated at 4° C overnight. One hundredmicroliters of 1: 10 goat antirabbit globulin in PBSthen were added, incubated at 37° C for 1 hour, andat 4° C for 4 hours.

Total radioactivity was then obtained in a Pack-ard autogamma scintillation spectrometer. The test

tubes were then centrifuged in a Sorvall SuperspeedRC 2-B automatic refrigerated centrifuge at 3500rpm (1500 g) for 30 mm at 4° C. The supernatantwas then suctioned off, and the precipitate wascounted in the gamma counter. Percentage bindingof 1251-rat albumin was then calculated, that is,

100 x count of precipitate — background —

count of total — background—

% bound.

A curve was constructed from the standard sam-ples. After obtaining the fraction of radioactivitybound in unknown samples, we determined albuminconcentrations from the standard curve. If the un-known experimental sample had a concentration of> 10 or < 0.1 jig/ml, the unknown experimentalsample was either further diluted or concentratedand the assay was repeated. The assay had a coeffi-cient of variation of 20%.

Lyophilized purified rat albumin and human albu-min and derivatized human albumin were standard-ized by weight. For rat and human albumin, the im-munoreactivity was related to fresh rat serum albu-min and fresh human serum albumin by determiningthe albumin present after measuring the total pro-tein with the Biuret reaction and the albumin frac-tion by electrophoresis. The lyophilized albuminvalues were corrected to the fresh serum values.

Statistical analysis. The results were studied bycovariance analysis [18]. All data, except for the al-bumin concentration, UA1bV, CAIb, and the clear-ance ratio for albumin (CA1b/CIfl x 100) were ex-pressed as arithmetic means. Previous observationsin our laboratory have shown albumin concentra-tions and UAIbV to be distributed normally logarith-mically [12]. The data pertaining to CAIb and GFRunderwent logarithmic transformation before analy-sis, and the data are expressed as geometric means.

The correlation coefficient of GFR, Crat Aib andChuman Mb were computed with the following covari-ance model to remove the effects of differences be-tween animals. For this analysis only those datapoints containing complete observations on both ratand human albumin were used. The model usedwas:

= j + bX + R, + S(1) + RT1k + ijk1

where is the overall mean; X, is the covariate (inthe control periods for each rat); b is the coefficientof the covariate; R1 is the treatment effect of the in-fusion of human albumin and the two species of al-tered human albumin; S(1) isjth rat tested within theith group; Tk is kth experimental time period; RTIk

Effect of p1 on renal handling of albumin 369

is treatment X time interaction; Cliki is error term(RST)(l)k interaction).

Differences between the treatment groups weretested by the Duncan test [18], with a level of signif-icance ofF = 0.01.

Results

Characterization of albumin derivatives. The im-munoelectrophoretic analysis of several albuminpreparations is shown in Fig. 1. The electrophoreticmigration of the cationized albumins was clearly al-tered; the albumin derivatives retained immuno-logic reactivity with the antialbumin serum. Withisoelectric focusing (Fig. 2), the majority of the ma-terial for the two albumin derivatives reported heredid not migrate as a single band; rather, as observedby Rennke et al in studies on ferritin and horse-radish peroxidase, there was a distribution rangingfrom 0.5 to 1 pH unit [3, 7]. No unaltered albuminwas found.

The physicochemical properties are summarizedin Table 1. Sodium dodecyl sulfate polyacrylamidegel electrophoresis showed that no significant po-lymerization of the albumin occurred as a result ofthe derivatization procedure. The Stokes radius ofthe two derivatized albumins (33 A) was slightlyless than that (35 A) of the unaltered albumin.

Results of infusion of albumin and albumin deriv-atives. The results of a representative experiment inwhich derivatized human albumin of p1 7.2 to 8.2

was infused are summarized in Table 2. Some of thephysiologic parameters measured are summarizedin Figs. 3 and 4.

Mean arterial blood pressure (MAP) was lowerthroughout the experiment in animals that receivedalbumin of p1 5.5 to 6.6. There was no difference inMAP in the other two groups of animals. MAP fellafter 3 hours in all groups. In animals infused withunaltered human serum albumin (p1, 4.9), urine vol-ume (V) increased during the last three experimen-tal periods, GFR decreased; fractional free waterreabsorption was relatively constant, and fractionalosmolar clearance and fractional sodium excretionincreased during the experimental period, and par-ticularly during the last two collection periods.Broadly speaking, most of these parameters be-haved similarly in animals infused with the two al-bumin derivatives of p1 5.5 to 6.6 and p1 7.2 to 8.2.

Serum levels, urinary excretion rates, and frac-tional clearances of rat albumin and of unalteredand altered human albumins are summarized in Fig.4. Unfortunately, the radioimmunoassay for humanalbumin was not sufficiently sensitive to detect thealtered urinary albumin in the third and fourth ex-perimental periods following infusion of the albu-min derivative, p1 5.5 to 6.5. The results of the co-variance analysis of the data on GFR and albuminclearances are summarized in Table 3.

There was no significant difference in GFR incontrol periods 1 and 2; nor was there a significant

Fig. 1. Immunoelectrophoresis using antihuman albumin. Well I contained normal human serum. The remaining wells contained 1 l ofprotein solutions (4 gIu1) as follows: 2, human albumin (Cutter); 3, cationized human albumin of p1 5.5 to 6.6; 6, cationized humanalbumin of p1 7.2 to 8.2; 7, cationized human albumin of p1 7.9 to 8.3; Wells 4 and 5 contained as yet uncharacterized preparations.

- ._-.: - •--- -

-. •4t.d'- -

fitV Ij

-:-. -'--.• I

370 Purtell et a!

EC0cx,

00

Fraction

Fig. 2. Isoelectric focusing pattern of altered albumin. Opticaldensity at 280 nm is the absorbancy of the fractions at 280 nm.Dotted line represents pH of each fraction. Material was iso-electric focused for 2 days in an ampholyte which formed agradient from pH 3 to pH 10. The major albumin fraction waspresent in fractions of pH 5.5 to 6.6.

variation with time during the four experimental pe-riods (Fig. 3 and Table 3). GFR was lower duringthe experimental than it was during the control peri-ods (F1,67 = 65, P < 0.0001). The fall in GFR wassomewhat greater after infusion of altered albuminthan it was after infusion of unaltered albumin, butthe differences were statistically significant (P <

Table 1. Some physical properties of the human albuminpreparations that were infused into ratsa

p1Molecular weight

daltonsStokes radius

A

4.9 69,000 355.5 to 6.6 69,000 337.2 to 8.2 69,000 33

a The p1 (isoelectric point) was measured by isoelectric focus-ing; molecular weight, by sodium dodecyl sulfate polyacrylamidegel electrophoresis; and Stokes radius, by Sephadex G-100 chro-matography.

0.01) only for animals infused with the more highlycationized derivative (p1, 7.2 to 8.2).

The concentration of rat serum albumin de-creased somewhat in the experimental period in allthree infused groups (Fig. 4), presumably associat-ed with the volume expansion. As expected, theconcentration in serum of the infused human albu-min and albumin derivatives decreased significantlyover the 105-mm following completion of the in-fusion. There was no difference in the slope of thecurves over the period of observation.

The rate of excretion of the several species of al-bumin (UAU)V) is also shown in Fig. 4. Of particularnote was the increase in excretion of endogenousrat albumin as well as of infused human albuminwhen the more highly cationized species (p1 of 7.2to 8.2) was infused. For the clearance of rat albu-min, there were no significant differences betweenthe two control periods (Table 3). In the three

Table 2. Experimental data on one animal infused with 40mg of albumin, p17.2 to 8.2"

MAP CTime Pe- mm V ml! SRA SHA U,,V UHAV

mm Events nods Hg 1d/min mm mg/mi mg/mi p.g/min p.g/min//

mmC,

j.d/min %

Cap.T%

C1CRA

Co,mx 100

C,fl

TcH,OxlOO

C,,, Fe1

Surgery0 Priming dose 3H inulin at 4.5 jiCi in 0.75 nI normal saline per 1.5 mm

Maintain 3H inulin at 3 Ci in 0.5 ml normal saline per hour60 Empty bladder

SC1 Cl 127 2.39 23.0UC1 3.82 1.32SC2 C2 130 0.91 25.0

0.06

0.07

0.0025

0.0077

1.1

2.7

0.9

2.3

0.13

0.47

120 UC2 3.76 1.77-+SE1 El 127 0.44 22.2 8.82UE1 4.61 4.61 193.6SE2 E2 122 0.88 22.5 4.46

0.21

0.38

21.96

34.25

0.022

0.043

4.99

3.86

104.6

90.1

4.6

3.3

3.5

1.8

1.12

1.80

180 UE2 13.29 8.64 152.8SE3 E3 110 1.14 21.5 2.58UE3 29.97 2.46 7.94 13.6SE4 E4 117 0.88 21.0

0.37

0.26

5.25

1.29

0.032

0.030

0.46

0.16

14.2

4.9

4.8

7.5

2.2

3.7

2.79

4.82

240 UE4 33.02 5.55 3.2Nephrectomy

a Abbreviations used are: MAP, blood pressure at left femoral artery; V, urine excretion rate; C,,,, inulin clearance; SRA, serum level ofrat albumin; SHA, serum level of human albumin; URAV, excretion rate of rat albumin; UHAV, excretion rate of human albumin; CRA, ratalbumin clearance; CHA, human albumin clearance; S, serum; C, control; U, urine; E, experimental; Cosm, osmolar clearance; TcH,O,free water reabsorption; FENa, fractional excretion of sodium; —*, infusion of 40 mg albumin. Body weight of rat at 0 mm was 248.3 g.

0.

0.5

0.4

0.1

S

U-(7

Effect of p1 on renal handling of albumin

LL(7Q0x

0r0

U-(700x00

371

groups as a whole, rat albumin clearance was great-er during the four experimental (mean, 0.0125 pumm) than it was during the two control (mean,0.0029 p1/mm) periods (F1,57 = 17; P <0.0001). Inthe experimental periods there was no significantchange in rat albumin clearance with time (F3,17 =0.77; P = 0.52), but there was a highly significantdifference in rat albumin clearances and clearanceratios between the three infusion groups (F2,9 =25.8; P < 0.0001). The within-group comparisonsshowed that the rat albumin clearances of the groupreceiving the albumin derivatives of p1 7.2 to 8.2differed significantly (P < 0.01) from those in boththe group receiving derivatized albumin with a p1 of

5.5 to 6.6, and the group receiving native albumin.The clearance ratio of rat albumin (CAIb/Cin)

changed little in the rats receiving unaltered humanalbumin (Fig. 4) (average, 0.0069%); that of the ratsreceiving derivatized human albumin of p1 5.5 to 6.6also varied little except in the first experimental pe-riod. In contrast, the clearance ratio of those ani-mals receiving albumin of p1 7.2 to 8.2 was in-creased 14-fold over the control values in these ani-mals. For the clearance of the infused humanalbumins (Table 3), there were clearly significantdifferences among the three groups (F2,9 = 34.3, P <0.0001). Within-group comparisons showed that theclearance of albumin of p1 7.2 to 8.2 was significant-

— —a——a

o.—o p1 4.9e-—.• p1 5.5-6.6•—--• p1 7.2-8.2

o-i—a-_.T'a' ._..-a- •-• •-

I I I

150

SS

a-

100

50

.8 30

>

10

8

8

4

8

4

4

24

zUJU-

,.

/

1 2 1 2 3 4 1 2 1 2 3 4

Control Experimental Control Experimental

Fig. 3. Summary of selected physiologic data on 5 rats infused (arrow) with normal human albumin p14.9 (O—O),4 rats infused withalbumin p1 5.5-6.6 (C----C), and 5 rats infused with altered albumin p1 7.2-8.2 (S-'-'-•).

372 Purtell et at

Table 3. Summary of results of covariance analysis'

Effect analyzed GFR Rat albumin clearance Human albumin clearance

Control periods I vs. 2 NS NS NSControl periods (1 + 2) vs. experimental periods F1,67 = 65.05 F1,57 = 17.02

(1+2+3+4) P<0.0001 P<0.0001Covariate (control vs. experimental periods) F1,10 = 6.31

P=0.03F1,9 = 9.16

P<0.01Treatment groups F3,19 = 7.49

P<0,01F2,9 = 25.85

P<0,0001F29 = 34.35

p<0.0001Within-group comparisons

p14.9vs.p15.5-6.6 NS NS Sp14.9 vs. p17.2-8.2 S S Sp15.5—6.6 vs. p17.2—8.2 S S S

Timebetweenexperimentalperiods F3, = 1.06P = 0.38

F317 0.77P = 0.52

F3,17 = 1.81P = 0.18

Treatment group x time interactions F6,33 = 1.58P = 0.19

F4,17 = 0.91P = 0.48

F4,17 = 4.07P = 0.02

a Differences are significant (5) or not significant (NS).21 Analyzed by the Duncan Test.

- Human

I

Human

IRat10.Or 80

I I

L 60r

St H1-2 I<40F.01 ,'-s I

I 0I 1..0 I I F

20

S

0.0011 lllll ______ 0121234 121234 121234 121234C E C E C E C E

Fig. 4. Summary of data on the handling of the infused human (HA) and endogenous rat (RA) albumins. Symbols are defined in Fig. 3.

100 Rat

I

0,0.CES

ES'I,

11)

- Human

II,/'

S

S

0

C0a,Uxa,=ES.0

10

1.0

0.1 ,.j,,iliiiii

Rat

I100

10

1.0

0.1

0.0111111

0—op1. 4.9e---epl, 5.5 to 6.65- "•pI, 7.2 to 8.2

ly greater (P < 0.01) than that of p1 5.5 to 6.6,which, in turn, was greater (P <0.01) than that ofunaltered human albumin. Within the four experi-mental periods as a whole, there was little dif-ference in the clearance of the human albumin spe-

cies (F3,17 = 1.81, P = 0.18), but there was somedifference in clearance with time among the groups;(F4,17 = 4.07, P = 0.02).

The geometric mean human albumin (CAIb/CIn)clearance ratio (Fig. 4) in the rats receiving native

Effect of p1 on renal handling of albumin 373

Table 4. Localization in the rat kidney of human albumin and of the altered human albuminsa

p1 of albumin infused

Glomeruli Tubules

Mesangium Basement membrane Bowman's capsule Droplets Basement membrane

4.9 0 0 0 0 05.5to6.6 0 0 0 07.2to8.2

Graded semiquantitatively on a scale from 0 to + + + +

albumin (0.0048%) was similar to that of endoge-nous rat albumin. The clearance ratio for altered hu-man albumin in rats receiving human albumin of p15.5 to 6.6 was much greater (0.013 and 0.011% in thefirst and second experimental periods). It was con-siderably higher than this in rats infused with al-tered albumin of p1 7.2 to 8.2 (0.74, 4.5, 0.55, and0.13% in the four experimental periods). When theclearances of the human and rat albumins werecompared, it was observed that the ratio Chuman Aib'Crat Ath was close to 1 for animals infused with unal-tered human albumin; it was 8 to 10 for those re-ceiving albumin of p1 5.5 to 6.6, and, in the initialperiods, following infusion of albumin of p1 7.2 to8.2, it was 19 to 64. The results of the immuno-fluorescence studies are shown in Table 4 and Fig.5. After infusion of albumin of low p1, human albu-min could not be detected. In those rats receiv-ing albumin of p1 5.5 to 6.6, there was equivocaldroplet formation in tubules. In the rats receivingalbumin p1 7.2 to 8.2, there was uptake of humanalbumin by the mesangium, 2+ staining of the gb-merular basement membrane and of Bowman's cap-sule, droplets in the tubules, and marked fluores-cence of the tubular basement membrane.

Discussion

It would have been ideal to infuse the normal andaltered human albumins at a constant rate. The dif-ficulties in making large enough amounts of materialavailable and in making measurements in urinewhen the concentrations are relatively low pre-cluded this. Studies with polysaccharide macromol-ecules of narrow size ranges were done with contin-uous infusion by Wallenius, who showed that in thedog neutral dextrans of molecular radius less than23 A had clearances approaching the glomerular fil-tration rate; that the clearance decreased sharply asthe molecular radius of the dextrans increased from23 to 34 A; and that for molecules whose radius ex-ceeded 34 A, the clearance was only a minute frac-tion of the gbomerular filtration rate [19]. Whenpolydisperse polysaccharide macromolecules areinfused, as has been done in recent years, the smallmacromolecules are excreted relatively rapidly, and

the large molecules relatively slowly. Thus, evenwhen constant infusions are being given, the plasmalevels must be changing during the experiment.

The results of the present experiments showclearly, despite a coefficient of variation of 20% inthe assays, that as the isoelectric point is increasedthe excretion rate of albumin increases by orders ofmagnitude. It is possible that the increase in clear-ance of the altered albumins is due to the slight de-crease in molecular radius from 35 A to 33 A. Asthe molecular size of both altered albumins was 33A and the more highly cationized derivative wascleared more rapidly, the slight decrease in molecu-lar size cannot be the sole explanation, and thecharge difference would appear to be important.Failure of tubular reabsorption alone cannot ac-count for the marked increase in excretion and frac-tional clearance of the albumin of p1 7.2 to 8.2, be-cause the difference in the clearance ratio of thisderivative as compared with unaltered albumin is atleast three orders of magnitude larger.

Table 5 is an attempt to summarize briefly thetypes of experiments that have been reported by in-vestigators seeking to study the permeability tomacromolecules of the glomerular capillary wall.For many years, experiments were done that ad-dressed the importance of the molecular weight orStokes radius of the macromolecule [20]. Variouspolysaccharide macromolecules, vegetable pro-teins, ferritin, and nonplasma proteins were used.Glomerular permeability was assessed physiologi-cally (using clearance measurements) or morpho-logically. The tracers used were chosen, not primar-ily because they were valid analogs of the major hu-man plasma protein albumin, but rather becausethey had certain characteristics, for example, elec-tron density, peroxidase activity, or absence oftubular reabsorption, that enabled them to be mea-sured.

Evidence that the glomerular basement mem-brane was a major barrier to filtration came fromferritin studies by Farquhar, Wissig, and Palade [4].An alternative view, that placed major importanceon the slit pore, came from the studies on proteinswith peroxidase activity by Graham and Karnovsky

374 Purtell et al

Fig. 5. A Immunofluorescent study of renal cortex of rat infused with altered human albumin of p1 7.2 to 8.2 showing linear staining ofglomerular basement membrane with fluorescein conjugated antisera to normal human albumin x 1320. B Immunofluorescence studyfrom same rat showing that material immunoreactive with antisera to human albumin is present both as droplets within tubular cyto-plasma and in tubular lumen (X320). C Section of outer medulla from same rat showing intense fluorescence of tubular membrane(x320).

Effect of p1 on renal handling of albumin 375

Table 5. Interpretative summary of experiments designed to examine the interrelationship between glomerular permeability and certaincharacteristics of protein and polysaccharide tracer moleculesa

Macromolecular tracer

Experimental approach Morphologic changes

Exogenous tracer Endogenousalbumincontrol

Footprocess

effacementGlomerularpolyanion

Bindingof tracer ReferencesMorphologic Physiologic

Molecular size, Stokes radiusUnaltered animal and vegetable proteins Yes Yes No Absent ND Yes 5, 6,8,21

ofmolecularradius 19to61 A (Ly) (Ly)(HRP) (HRP)

(Ly,CATLAC)

22, 23

Dextrans Yes Yes No None ND None 19, 20Polyion charge

Dextrans No Yes No ND ND ND 1, 2Systematically altered animal and

vegetable proteins (FER, CAT, HRP) Yes Yes No None ND Yes, ifp1>7

3, 7

Systematically altered albumin Yesb Yes Yes Yesb Presentb Yes This study

a Abbreviations used are: CAT, catalase; FER, ferritin; HRP, horseradish peroxidase; LAC, lactoperoxidase; Ly, lysozyme; ND, notdone.

b Clyne et al, unpublished observations.

[6] and Graham and Kellermeyer [8]. These studieswere done before it was appreciated that the electri-cal charge on a macromolecule might also be impor-tant [1, 2, 21]. As this concept gained acceptance, itwas realized that most of the tracer proteins withperoxidase activity had apT > 7, and they were thenshown to be capable of binding to negativelycharged structures in the glomeruli [7, 21]. Presum-ably, such binding may itself result in change in thefunctional integrity of the glomerulus.

Lysozyme (p1, 11), which has been used exten-sively in physiologic studies [22], has been shownalso to bind extensively to negatively charged struc-tures in glomeruli and tubules [23]. Such extensivebinding makes it very difficult to interpret the mean-ing of clearance measurements with this protein.Alterations in the glomerular integrity to macromol-ecules may presumably be reflected in alterations inthe handling of endogenous plasma proteins andparticularly albumin, but this was not studied in anyof the experiments described.

Table 5 also summarizes the experiments donewith a view to examining the influence of electricalcharge on the macromolecule. Studies with dex-trans showed the influence of charge on their trans-port, but were limited to clearance measurements.Recently, several proteins, that are either electron-dense or have peroxidase activity, have been modi-fied to produce protein species with various isoelec-tric points [3, 7]. Clearance studies have shown thatpositively charged proteins are more readily trans-ported through the glomerular capillary wall; mor-phologic studies, that cationic protein may bind toglomerular structures.

The present study is, to our knowledge, the onlystudy in which an important endogenous and analo-gous protein has been studied, namely the animal'sown albumin. The data show that increasing theisoelectric point of the heterologous albumin causednot only an increase in heterologous albumin excre-tion, but also increased nephron permeability to na-tive endogenous albumin. This implies that achange in the functional structure of the nephronhas occurred. Immunofluorescent microscopicstudies show binding of the highly cationized albu-min in the glomerulus, probably to sialoprotein.Studies in our laboratory, to be reported separately,also show focal effacement of foot processes (Clyneet al, in preparation).

Acknowledgment

This work was supported by grants AM 17196,AM 17330, and AM 18466.

Reprint requests to Dr. Amadeo J. Pesce, University of Cin-cinnati Medical Center, Division of Nephrology, 3412 College ofMedicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267, USA

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