Isolation and characterization of rat tubular basement membrane

Kidney International, Vol. 12 (1977), pp. 238—243

Isolation and characterization of rat tubular basement membrane ISTVAN KRIsK0, ERICA DEBERNARDO AND CLIFORD S. SATO Research Service, Veterans Administration Hospital, and Departments of Pharmacology and Pathology, Baylor College of Medicine, Houston, Texas

Only a limited knowledge of the structure and

Isolation and characterization of rat tubular basement membrane. Tubules were isolated by the passage of coarsely homogenized kidney parenchyma through a series of stainless steel sieves of varying

function of the tubular basement membrane (TBM) of the kidney is available, in contrast to the glomerular basement membrane (GBM), whose structure and

pore sizes. The isolated tubules, which were free of glomeruli or any contaminating tissue, based on light microscopic examination, served as starting material for the isolation of tubular basement membrane (TBM). The tubules were sonically disrupted, and the subsequent isolation procedure was conceptually similar to that of the isolation of glomerular basement membrane (GBM). The chemical composition of TBM was compared to that of GBM and collagen. 3-hydroxyproline, a marker for basement membrane protein, was present in higher amount in TBM (10 residues per 1,000 amino acid residues) than in GBM (7 residues per 1,000). Glycine and hydroxylysine content was also higher in TBM (255 and 27 residues per 1,000, respectively, in contrast to 204 and 18 residues in GBM). The total carbohydrate content was 7.6 mg per 100 mg of dry weight (neutral sugars, 6.3; GleN, 0.9; GaIN, 0.1; and sialic acid, 0.3). By electron microscopic examination, the isolated TBM was similar to GBM, except for greater variability in diameter and serrated appearance of one surface of the tubular basement membrane fragments. The method is suitable for the isolation of TBM

function are well characterized [1—11]. Our interest in

the chemical composition of IBM has been stimulated by two factors: our previous work on glomeruli and GBM [12—15], and the steadily growing evidence that IBM is involved in renal diseases [16—251. Knowledge of the chemical composition of IBM should be helpful in understanding its function and its involvement in disease. Single tubular segments have been isolated previously and used to great advantage in physiologic studies [6, 26]. Single segments of IBM, however, will not suffice for detailed chemical studies. Considering anatomical and histologic relationships within the whole kidney, it is implicit

in sufficient quantity for subsequent chemical analyses and immunologic studies.

that a tubular preparation that is free of other contaminating tissue elements be used as starting material for the isolation of TBM of highest purity. Furthermore, for adequate chemical analysis, IBM has to be isolated in sufficient quantity. Human IBM has been previously isolated and studied by Mahieu and Winand [8], and Sato et al [10]; bovine IBM, by Ferwerda et al [9]; and rabbit IBM, by Meezan et al [11]. There is no published work, however, on the study of isolated rat IBM.

Isolement et caractérisation de Ia membrane basale tubulaire du rat. Des tubules ont été isolés par passage d'une homogénat gros-

sier de parenchyme renal a travers une sCrie de tamis, en acier inoxydable, de diffCrentes tailles de pores. Les tubules isolés, qui ne

sont contaminés ni par des glomérules ni par d'autres tissus a l'examen microscopique, ont servi de materiel pour l'isolement de Ia membrane basale tubulaire (TBM). Les tubules ont été traitCs par les ultra-sons et l'isolement ultérieur a été semblable, dans sa conception, a celui des membranes basales glomérulaires (GBM). La composition chimique de TBM a été comparée a celle de GBM et au collagene. La 3-hydroxy-proline, un marqueur des protéines de Ia membrane basale, existe en plus grande quantité dans TBM (10 résidus pour 1,000) que dans GBM (7 rCsidus pour 1,000). La glycine et l'hydroxylysine sont aussi plus abondantes dans TBM (255 et 27 résidus pour 1,000 respectivement, a comparer a 204 et

Besides, the above workers either employed different techniques for the isolation or the chemical analysis has been incomplete in some respects. For instance, the human IBM analyzed by Mahieu and Winand [8] was apparently contaminated by interstitial collagen, and Sato et al [10] and Meezan et al [11] did not determine 3-hydroxyproline content. This publication describes the isolation of rat kidney tubules and IBM, as well as the chemical composition of TBM. The isolation method offers IBM of sufficient purity and in ample quantity for meaningful chemical analyses.

18 résidus dans G BM). Le contenu total en hydrates de carbone est de 7, 6mg pour 100mg de poids sec (sucres neutres 6, 3; GIN 0,9;

GaIN 0,1; et acide sialique 0,3). A l'examen en microscopie électronique, les TBM isolées sont semblables a GBM exception faite d'une plus grande variabilité du diamètre et de l'aspect dentelé d'une des surfaces des fragments de TBM. Cette méthode est adequate pour l'isolement de TBM en quantité suffisante pour les analyses chimiques ultérieures et les etudes immunologiques. Received for publication October 18, 1976; and in revised form May 23, 1977. 1977, by the International Society of Nephrology.

238

239

Isolation of tubular basement membrane

Methods

Isolation of renal tubules and glomeruli. The tech-

nique of isolation was conceptually similar to that used for the isolation of kidney glomeruli [1, 12, 13].

Approximately 80 Sprague-Dawley rats, weighing 150 to 250 g, were used for each experiment. Under ether anesthesia, the animals were exsanguinated, the kidneys removed, cut into four pieces along two per-

sonicate by low-speed centrifugation (IEC, l7OX g for one minute) and discarded, while the supernatant was centrifuged at 3,000X g for 20 mm (pellet A). Pellet A was resuspended in approximately 40 ml of 1M sodium chloride. Extreme care was used to effect complete suspension by vigorous stirring with a glass

rod, and the centrifugation (3,000X g for 20 mm) was repeated; this was designated the first wash.

pendicular planes, and the papillae and grossly

Washing was repeated six additional times in IM sodium chloride, and four times in distilled water. The final pellet was designated "IBM." Approximately

wise.

pooled kidney tissue (wet weight). Aliquots of TBM were processed for electron microscopic examination, and the remaining material was lyophilized for subsequent chemical analyses. Other procedures and materials. For amino acid

observable fatty tissue of the hilum discarded. After the removal of the kidneys, subsequent procedures were performed at 0 to 4°C, unless specified other-

The quadrisected kidneys were minced coarsely with a razor blade and placed on a stainless steel sieve with a pore size of 600gm (28-mesh, in Tyler equiva-

lents; all sieves were custom-made by W. S. Tyler, Inc. and purchased via Fisher Scientific, Houston,

TX). The tissue was carefully forced through the sieve with the bottom of a small beaker, using ample amounts of ice-cold physiologic saline, the latter in

order to "wash through" and suspend the freed tubules and glomeruli. The fibrous material remaining on top of the sieve was discarded. That portion which passed through this sieve, containing the tubules and glomeruli (as well as contaminating tissue fragments

and individual cells), was passed successively (by

force of gravity alone) through the 250-itm, the l80-tm, and finally, through the l25-ttm pore-size

sieves. Tubular fragments of varying sizes remained on top of the l25-m sieve, whereas glomeruli passed through this sieve. The tubules were suspended in saline in 50-mI plastic tubes and centrifuged at 170X g for three minutes in a refrigerated centrifuge (IEC PR-J). The final pellet was designated "tubular fraction." The material which passed through the sieve of

l25-zm pore-size contained the glomeruli, which were isolated as described previously [13]. Isolation of TBM and GBM. The method previously employed for isolation of GBM [1, 13] was adapted for the isolation of IBM. The tubules were suspended in approximately four volumes of 1M sodium chloride solution and sonicated, using a Biosonik IV apparatus (Bronwill-VWR Scientific, Hous-

ton, Ix). A probe with a 20-mm diameter was

employed with 90% power intensity. Fourteen 30-sec bursts were applied with cooling of the probe in ice-

water between successive bursts of sonication. It is our impression that more energy is required for the sonic disruption of tubules than that of glomeruli. Even after the seven-minute sonication, some of the material withstood attempts at disruption, and "fibrous" particles were visible to the naked eye. These fibrous particles were separated from the rest of the

200 mg of IBM could be isolated from 200 g of

analyses, samples were hydrolyzed in 6N hydro-

chloric acid at 110°C in vacuo for 22 hr, and amino acids were determined using an amino acid analyzer (Beckman, model l2OC). Amino sugars were determined after hydrolysis of the material in 6N hydrochloric acid at 100° C for 6 hr in vacuo, employing the basic column of the amino acid analyzer at a pH of 5.28 and at 55°C. (Under the conditions used, the recovery of GlcN and GaIN was 95 and 93%, respectively; the values given were corrected accordingly.) Soluble collagen was isolated from rat skin by a method essentially as described by Piez et al [27]. For the determination of neutral sugars, samples were hydrolized in 2N sulfuric acid at 100°C for four hours in sealed tubes. Total neutral sugars were determined by the anthrone reaction [28], using glucose as standard. Prior to the anthrone reaction, amino acids and

peptides were removed from the hydrolysates by passing them through coupled Dowex 50—X4 and Dowex l—X8 columns [1, 13]. Sialic acid was determined by the method of Warren [29]. Electron microscopic procedures. Samples of IBM and GBM were fixed in 4% glutaraldehyde, followed by post-fixation in 1% osmic acid, and processed for electron microscopy in the usual way [30]. Results

During the preparation of both tubules and glomeruli, the isolation procedure was monitored closely by,

light microscopy. The unstained isolated kidney tubules are shown in Figure 1. In the tubular preparation, neither recognizable glomeruli nor interstitial tissue contamination were present; in turn, the gbmerular preparation contained neither tubules nor any other contaminating material. The starting mate-

rial for the isolation of TBM was preparations of tubules as they appear in Figure 1. (A photomicro-

240

Krisko et al

per 1,000 amino acid residues, respectively; 3-

hydroxyproline was not detected in soluble rat skin collagen with the procedures used; b) the presence of large amounts of 4-hydroxyproline in all three glycoproteins; c) the glycine content of TBM being higher (255 residues per 1,000) than that of GBM (204 resi-

dues per 1,000); in this respect, TBM is closer to collagen than GBM is; d) both IBM and GBM containing half-cystine, whereas mature collagen does not; and e) as compared to collagen, GBM containing approximately four times more, and IBM five times more, hydroxylysine. The total neutral sugar content of isolated TBM was 6,3 mg/100 mg of dry weight of the membrane

(Table 2). This value is similar to that found for GBM. Furthermore, glucosamine, galactosamine, and sialic acid are present in TBM in approximately the same proportion as in GBM (Table 2). Discussion

Fig. 1. Isolated rat kidney tubules. Note that several loops of tubules are folded on each other (magnification, X 200).

We have adapted procedures previously employed in the study of glomeruli and GBM for the isolation of tubules and TBM from rat kidneys. Tubules were isolated by passage of the coarsely homogenized kidney parenchyma through a series of stainless steel sieves of varying pore sizes. In order to isolate TBM, the tubules were sonically disrupted; the subsequent isolation procedure was conceptually similar to that Table 1. Amino acid composition of rat tubular basement

graph of glomerular preparations employed as starting material for the isolation of GBM has been published already; cf. Ref. 13, Fig. 1.)

An electron micrograph of the isolated TBM is presented in Figure 2. Note the varying diameter and

serrated appearance of one surface of some of the tubular basement membrane fragments. It is our impression that TBM preparations show greater variability in thickness than the variation in thickness in corresponding GBM preparations (not shown). The greater variability in thickness of TBM preparations may be related to the various segments of the tubular apparatus. We point out, however, that this is only a hypothesis, since the procedure for the isolation of

TBM does not allow separation of TBM fractions originating in different segments of the tubular apparatus. The TBM does not show any significant structural organization at this magnification. The amino acid profiles of TBM, GBM and soluble rat skin collagen are presented in adjacent columns of

Table I in order to compare more easily the similarities and differences. The following are considered significant: a) a somewhat higher 3-hydroxyproline content in TBM than that of GBM, 10 vs. 7 residues

membrane (TBM), glomerular basement membrane (GBM), and skin collagen Residues, per 1.000 amino acid residue? Amino acid 3-Hydroxyproline 4-Hydroxyproline Aspartic acid Threonineb Serine5

Proline Glutamicacid Glycine Alanine Valine HaIf-cystine Methionine Isoleucine Leucine

Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine

TBM

GBM

10.4 + 1.0 81.6 + 4.1 61.6 1.9

7.0 + 1.2 59.4 + 1.7 71.8 1.5 42.5 + 2.3 54.4 + 1.4 67.9 + 2.6 99.6 1.4

33.! + 2.8 47.7 1.8

64.5 2.6 91.8 + 2.1

255.6 8.3 51.5 2.9 35.1 + 2.0 17.7 + 4.0 14.5 + 0.7

204.1 54.9

5.2 0.7 0.8

40.7 36.7 + 4.5 13.9 + 0.5 28.3 + 0.7 64.4 + 1.4

Collagen

— 92,1 + 1.5 45.1 0.7 19.7

1.2

0.4 114.8 + 1.0 71.5 0.5 37.1

354.2 104.5 22.5

1.2

0.4

0.9

1.5

29.4 0.4

6.0 0.2 10.6 0.2 24.4 + 0.1 1.8 1.2

26.8 + 3.3

18.8 + 1.5 22.5 + 0.5

11.1 + 0.2 5.3 0.6 26.3 2.2

27.3

1.2

57.2 2.8 15.9 + 1.2 29.0 19.9

15.8

1.2 1.5

43.3 + 2.3

a Values are mean + SD. N = 4,

19.0

19.3

0.6

3.5

1.0

46.2

3.8

49.8

I.!

where N denotes the number of

experiments (approximately 80 rats were used for each experiment).

Not corrected for losses during hydrolysis. None detected.

Isolation of tubular basement membrane

241

Fig. 2. Electron micrograph of isolated tubular basement membrane from rat kidneys. Some of the IBM fragments show serrations on one side, and there is great variability in their diameter (magnifIcation, X 41,200).

analyses of isolated rat IBM revealed that: a) IBM is

tine, than GBM. But, unlike GBM, one surface of most of the IBM fragments has a serrated appear-

a glycoprotein with approximately eight percent

an ce.

of the isolation of GBM. Detailed compositional carbohydrate content (based on dry weight), and it is similar in composition to GBM; b) 3-hydroxyproline, a marker of basement membrane protein, is present in a slightly higher amount in TBM (10 residues per I ,000 amino acid residues) than in GBM (7 residues

per 1,000); and c) IBM contains more 4-hydroxyproline, glycine, and hydroxylysine, but less half-cysTable 2. Carbohydrate composition of rat tubular basement membrane

Amounta Carbohydrate Total neutral sugarsb Glucosamine Galactosamine Sialic acidc

mg/IOO mg of dry weight

6.29

0.52

0.94 0.07 0.10 + 0.01 0.32 0.03

Values are mean + SD (N = 4, where N denotes the number of experiments). Individual neutral sugars were not determined. Expressed as glucose equivalents. N-acetylneuraminic acid was used as a standard.

Notwithstanding the great deal of effort expended in the study of GBM, thus far there has been only a relatively small amount of work devoted to the study of IBM. We are not aware of any previous report on the chemical composition of rat IBM. (Misra [31] published an abstract in which he mentioned, without giving specific data, the isolation of rat TBM.) The

isolation of TBM from species other than rat has already been reported, the first one by Goodman, Greenspon, and Krakower [32]. Their primary aim was to study the antigenicity of various elements of the canine kidney. They dissected out special areas,

the so-called cortex corticis, which were free of glomeruli, and which they sonicated and washed in 8% sodium chloride. Although sufficient data are not given to enable one to formulate a reliable opinion on the precise composition of the isolated substance, it

was known to them that their material contained hydroxyproline, and that it was related to collagen. Mahieu and Winand [8] were the first to report the isolation of human TBM and also the first to publish

242

Krisko et a!

detailed compositional analyses of any TBM. Their isolation procedure differed from ours in several important points. Tubules were suspended in isotonic saline, rather than I M sodium chloride, and a homogenizer (Potter-Elvehjem) was used for disruption, rather than sonication. Their own electron micrographs show contamination of TBM by interstitial

collagen, although their GBM preparation was of good quality. The decreased 3-hydroxyproline, halfcystine and hydroxylysine content was added evidence of contamination by interstitial collagen.

We note that in addition to IBM we have also isolated another fraction from tubules which contained significant amounts of "contaminating" interstitial collagen (decreased 3-hydroxyproline content and the presence of cross-striations under electron microscope; data not given under Results); whereas we have never detected interstitial collagen in sonicated material derived from isolated glomeruli. We hypothesize that peritubular fibroblasts, intimately associated with the tubules, were the source of this

collagen. Therefore, great care must be exercised

when preparing TBM, even though apparently "pure" renal tubules are available as starting mate-

high value of 3-hydroxyproline in TBM deserves spe-

cial emphasis. The marked difference in content of

this amino acid and 4-hydroxyproline between known basement membranes and interstitial collagen allowed Man and Adams [34] to estimate total base-

ment membrane in whole animals by analysis for total 3-hydroxyproline and 4-hydroxyproline. Taken together, the above data can be viewed as evidence that 3-hydroxyproline is a marker for basement membrane glycoproteins.

Significant advances have been made in understanding the complex function of renal tubules and the relationship of anatomical structure to their function [6, 7, 26, 35—37]. Little is known, however, of the function of IBM per Se, especially when compared to GBM [38]. An issue of special significance in consid-

ering renal diseases is the immunologic cross reactivity between IBM and GBM. The availability of isolated and purified preparations of both these basement membrane glycoproteins should be helpful in

obtaining answers to this important question. Detailed knowledge of the chemical composition is therefore not only of intrinsic interest, but also of potential physiologic importance.

rial.

TBM is closely related to GBM, yet compositional differences exist which delineate these two glycoproteins from one another. It is recognized that data on amino acid composition may vary from preparation to preparation and according to the method of isola-

tion of the basement membrane (e.g., duration and energy of sonication [33]). Because of the foregoing, the significance of the observed differences in amino acid composition between TBM and GBM can be questioned. Nevertheless, on the basis of the data presented in this paper, it appears that TBM and GBM—although closely related—are unique and distinguishable glycoproteins. The definitive answer to

this important question, however, will come only

Acknowledgments

This work was supported by Veterans Administra-

tion research grants (Project No. 12 38-01 and Project No. 2015-01). A preliminary report of this work appeared in abstract form (Lab Invest 32:450, 1975). Dr. C. C. Tisher gave encouragement

and helpful suggestions; Dr. I. Daskal assisted in preparing the electron micrographs; and Miss K. Lewis and Messrs. S. Gergely and J. Putch gave technical help. Reprint requests to Dr. Is Wan Krisko, Suite 601, 1665 Palm Beach Lakes Blvd., West Palm Beach, Florida 33401, U.S.A.

when the total amino acid sequence of the constituent

peptide chains of both TBM and GBM becomes available. TBM, like other basement membranes, possesses

the following distinguishing characteristics when compared to interstitial collagen: a) the presence of 3-

hydroxyproline (10 residues per 1,000 amino acid residues, which is even higher than that of GBM, whereas interstitial collagen of vertebrates contains only trace amounts [34]); b) high hydroxylysine content (27 residues per I ,000; i.e., five times higher than that of collagen); c) high carbohydrate content (approximately eight percent of dry weight); and finally, d) the characteristic cross-striations of interstitial collagen are absent in IBM. Of the above properties, the

References 1. SPIRO RG: Studies on the renal glomerular basement mem-

brane. J Biol Chem 242:1915—1919, 1967

2. KEFALIDES NA: Chemical properties of basement membranes. mt Rev Exp Pathol 10:1—39, 1971

3. RHODIN JAG: Structure of the kidney, in Disease of the Kidney, edited by STRAUSS MB, WELT LG (2nd ed). Boston, Little Brown and Company, 1971, vol. 1, pp. 1—30 4. BERLINER RW: Outline of renal physiology, in Diseases of the Kidney (2nd ed.) edited by STRAUSS MB, WELT LG, Boston, Little Brown and Company, 1971, vol. 1, pp. 31—85 5. CAULFIELD JP, FARQUHAR MG: The permeability of glomeru-

Jar capillaries to graded dextrans: Identification of the basement membrane as the primary filtration barrier. J Cell Biol 63:883—903, 1974

243

Isolation of tubular basement membrane ment membrane in human renal allografts. mt

6. IMAI M, KOKKO JP: Transtubular oncotic pressure gradients

and net fluid transport in isolated proximal tubules. Kidney

mt 6:138—145, 1974 7. WELLING LW, GRANTHAM JJ: Physical properties of isolated

Antitubular basement membrane antibodies after renal transplantation. Transplantation 18:447—452, 1974 23. LEHMANDH, MARQUARDT H, WILSON CB, DIXON FJ: Speci-

ficity of autoantibodies to tubular and glomerular basement membranes induced in guinea pigs. J Immunol 112:241—248,

glomerular basement membranes of human kidney. Eur J Biochem 12:410—418, 1970 9. FERWERDA W, MEUER JFM, VAN DEN EIJNDEN DH, VAN DIJK W: Epithelial basement membrane of bovine renal tu-

buli. Hoppe Seylers Z Physiol Chem 355:976—984, 1974 10. SATO T, MUNAKATA H, YOSHINAGA K, YOSIZAWA Z: Com-

parison of the chemical composition of glomerular and tubu-

lar basement membranes obtained from human kidneys of diabetics and non-diabetics. C/in Chim A cIa 61:145—150, 1975 II. MEEZAN E, HJELLE iT, BRENDEL K, CARLSON EC: A simple

versatile, nondisruptive method for the isolation of morphologically and chemically pure basement membranes from several tissues. Life Sci 17:1721—1732, 1975 WG: Protein synthesis in kidney glomeruli. C/in Res 20:599, 1972 13. KRIsK0 I, WALKER WG: Protein and glycoprotein synthesis by isolated kidney glomeruli. Proc Soc Exp Bio/ Med

12. KRISKO I, WALKER

146:942—947,

1974

1974

24. LEHMAN DH, WILSON CB, DIxON FJ: Interstitial nephritis in

rats immunized with heterologous tubular basement membrane. Kidney mt 5:187—195, 1974 25. BARAEAS AZ, LANNIGAN R: Induction of an autologous immune-complex glomerulonephritis in the rat by intravenous injection of heterologous anti-rat kidney tubular antibody. Br J Exp Pathol 55:47—55, 1974 26. BURG M, GRANTHAM J, ABRAMOW M, ORLOFF J: Preparation

and study

Symposium: Energy, Biosynthesis and Regulation in Molecular Biology, edited by RICHTER D, New York, Walter de Gruyter Co, 1974, pp. 345—358

of

fragments of single rabbit nephrons. Am I

Physiol 210:1293—1298, 1966 27. PIEz KA, EIGNER EA, LEwis MS: The chromatographic separation and amino acid composition of the subunits of several collagens. Biochemistry 2:58—66, 1963 28. RoE JH: The determination of sugar in blood and spinal fluid with anthrone reagent. I Biol ('hem 2 12:335—343, 1955 29. WARREN L: The thiobarbituric acid assay of sialic acids. J

14. KRIsK0 I, GYORKEY F: Studies on the biosynthesis and struc-

ture of renal glomerular basement membrane, in Lipmann

Allergy

STICKEL DL:

perfused renal tubules and tubular basement membrane. I C/in Invest 51:1063—1075, 1972 8. MAHIEU P, WINAND RJ: Chemical structure of tubular and

Arch

AppI Immunol 45:675—689, 1973 22. WILSON CS, LEHMAN DH, MCCOY RC, GUNNELLS JC JR.

30.

Biol Chem 234:1971—1975, 1959 FARQUHAR MG, WIssIG SL, PALADE GE: Glomerular per-

meability: 1. Ferritin transfer across the norlnal glomerular capillary wall. I Exp Med 113:47—66, 1961

WG: Biosynthetic studies with isolated

31. MISRA RP: Glomerular and tubular basement membrane chemistry in experimental renal diseases. Fed Proc 32:827,

Biophys Ada 446:506—5 14, 1976 16. KLASSEN J, ANDRES GA, BRENNAN JC, MCCLUSKEY RT: An

32. GOODMAN M, GREENSPON SA, KRAKOWER CA: The anti-

IS. KRIsK0 I, WALKER

kidney glomeruli. Biochim

1973

genie composition of the various anatomic structures of the canine kidney. J Immunol 75:96—104, 1955

immunologic renal tubular lesion in man. C/in Immunol Immunopathol 1:69—83, 1972

33. KEFALIDES NA: Structure and biosynthesis of basement mem17. GERMUTH FG JR, RODRIGUEZ E: Immunopatho/ogy of the branes. Int Rev Connect Tissue Res 6:63—107, 1973 Renal Glomeru/us. Boston, Little Brown and Company, 1973, 34. MAN M, ADAMS E: Basement membrane and interstitial collapp. 1—227 gen content of whole animals and tissues. Biochem Biophys 18. BALDWIN DS, LEVINE SB, MCCLUSKEY RT, GALLO GR:

Renal failure and interstitial nephritis due to penicillin and methicillin. N Eng/ J Med 279:1245—1252, 1968 19. BORDER WA, LEHMAN DH, EGAN JD, SASS HJ, GLODE JE, WILSON CB: Antitubular basement-membrane antibodies in

methicillin-associated interstitial nephritis. N Eng/ J Med 291:381—384, 1974 20. KOFFLER D, SANDSON J, CARR R, KUNKEL HG: Immunologic

studies concerning the pulmonary lesions in Goodpasture's syndrome. Am I Pathol 54:293—305, 1969 21. KLASSEN J, KANO

K,

MILGROM F, MENNO AS, ANTHONE S,

Res Comm 66:9—16, 1975 35. TISHER CC, KOKKO JP: Relationship between peritubular

oncotic pressure gradients and morphology in isolated proximal tubules. Kidney mt 6:146—156, 1974 36. ROCHA AS, KOKKO JP: Permeability of medullary nephron segments to urea and water: Effect of vasopressin. Kidney ml 6:379—387, 1974 37.

SCHWARTZ

MM,

VENKATACHALAM MA: Structural differ-

ences in thin limbs of Henle: Physiological implications. Kidney mt 6:193—208, 1974 38. FARQUHAR MG: The primary glomerular filtration barrier—

ANTIIONE R, SEPULVEDA M, ELW000 CM, ANDRES GA:

basement membrane or epithelial slit? Kidney

Tubular lesions produced by autoantibodies to tubular base-

1975

mt 8:197—211,