Kidney International, Vol. 15 (1979), pp. 20—32

Biosynthesis of basement membrane matrix by isolated rat renal glomeruli J. THOMAS HJELLE, EDWARD C. CARLSON, KLAUS BRENDEL, and ELIAS MEEZAN Departments of Pharmacology and Anatomy, University of Arizona Health Sciences Center, Tucson, Arizona

Biosynthesis of basement membrane matrix by isolated rat renal

érulaire. La synthèse de membrane basale est inhibée par les

glomeruli. The incorporation of radioactive precursors into the extracellular basement membrane matrix has been investigated in a purified preparation of isolated rat kidney glomeruli. Using deoxycholate extraction of isolated glomeruli which were incubated with radioactively labeled amino acids and carbohydrates, as an assay system for the measurement of incorporation of basement membrane precursors into intact deoxycholate insoluble basement membrane material, we demonstrated the in vitro biosynthesis of this structure. The assay system minimized the possibility of contamination of the isolated labeled basement membrane with labeled cell membrane fragments which may occur with the standard isolation procedures involving sonication and centrifugation. A linear incorporation of labeled proline, lysine, glycine, glucosamine, and galactose into glomerular basement membrane was shown. Basement membrane synthesis was inhibited by metabolic poisons and protein synthesis inhibitors as well as by inhibitors of collagen synthesis, but not by colchicine, an inhibitor of collagen secretion. The appearance of '4Chydroxyproline in the basement membrane matrix was negligible during the first 4 hours of incubation and rose only to 1% of the total proline counts thereafter. The results are consistent with

poisons metaboliques et les inhibiteurs de Ia synthése protèique mais pas par Ia colchicine, un inhibiteur de Ia secretion de collagéne. L'apparition de 14C-hydroxyproline dans Ia matrice de la membrane basale est negligeable durant les 4 premieres heures d'incubation et n'atteint que 1% de la proline totale ensuite. Les résultats sont en faveur de Ia definition de Ia synthèse et de la deposition de membrane basale glomerulaire comme un système

a deux composants incluant un ou des composants glycoprotéine(s) non collagènes rapidement synthetises et déposés et un composant collagène qui est déposé seulement aprês un délai de 4 a 6 heures.

Basement membranes are specialized extracellular support structures that serve the additional important function in the kidney of being a continuous barrier between blood and urine in the glomerular capillaries. The basement membranes define the three-dimensional architecture of the renal glomeru-

the characterization of glomerular basement membrane syn-

lus even in the absence of cellular elements [1, 2] and may be responsible for directing the orderly repopulation of new cells in areas of focal injury where tissue repair is occurring [3]. The renal gbmerulus is the site of numerous disease-related processes which result in glomerular basement membrane alterations [4], foremost among which are the abnormalities of basement membrane structure and function as a result of diabetes mellitus, which are the most serious pathologic consequences of this disease [51. For these reasons and because of the relative ease with which kidney glomeruli can be isolated with sieving techniques [6, 7], glomerular basement membrane structure [7-11] and metabo-

thesis and deposition as a two component system comprising a rapidly synthesized and deposited noncollagenous glycoprotein component(s) and a collagenous component which is only deposited after a delay of 4 to 6 hours. Biosynthèse de Ia matrice de Ia membrane basale par le glomérule isolé de rat. L'incorporation de précurseurs radioactifs dans la matrice de la membrane basale extracellulaire a été étudiée

dans une preparation purifiée de glomérules isolés de rat. L'utilisation d'extrait par le déoxycholate de glomérules isolés incubés avec des acides aminés et des hydrates de carbone marqués comme système de mesure de l'incorporation des précurseurs de la membrane basale par la fraction insoluble dans le deoxycholate de la membrane basale a permis de démontrer Ia biosynthése in vitro de cette structure. Le système de mesure minimise Ia contamination possible de la membrane basale isolée marquee par des fragments de membrane cellulaire marques qui

peuvent exister du fait des procédés courants d'isolement qui impliquent les ultra-sons et la centrifugation. Une incorporéation

lism [12—20] have been the subject of extensive investigation. Among the problems involved in such studies are the preparation of highly purified meta-

linéaire de proline, lysine, glycine, glucosamine, et galactose marques a ete mise en evidence dans Ia membrane basale glom-

bolically active glomeruli, free of tubular con0085—253817910015—0020 $02.60

tamination, and the isolation of purified basement membranes free of contamination by cell membrane fragments, which are an obligatory product of the

© 1979 by the International Society of Nephrology

sonication techniques used in their isolation [11, 21—

Received for publication November II, 1977 and in revised form May 8, 1978.

20

Biosynthesis of glomerular basement membrane

221. Both of these problems are magnified in biosyn-

thetic studies of basement membrane synthesis in isolated glomeruli. In this system, contamination of the glomerular basement membrane with its slow

21

370 C in 4 ml of incubation buffer in a shaking water bath.

tabolism and a basement membrane of their own may make the unequivocal demonstration of the

Basement membrane isolation and analysis. After various times of incubation, aliquots of the incubation mixture were placed in 1 .5-ml microfuge tubes and centrifuged (Beckman Microfuge) for 30 sec. The supernatants were removed with a drawn pipet, after which the pellets were each twice sus-

biosynthesis and deposition of basement membrane

pended in 1 ml of saline and centrifuged. With vigor-

components into the isolated extracellular matrix

ous vortexing, 1 ml of 4% sodium deoxycholate

difficult to achieve. In the present investigation, we have minimized these problems by coupling the use

with 0.0 1% sodium azide was added to each saline-

turnover [11, 23—251 by cellular components with a more rapid turnover and by tubules with a high me-

of a procedure to isolate metabolically active rat renal glomeruli of greater than 99% purity [261 with

an assay based on a novel procedure for the isolation of ultrastructurally intact and chemically defined glomerular basement membrane [1, 27, 28] to demonstrate the incorporation of basement membrane precursors into the purified extracellular matrix. Methods

washed pellet. After standing overnight at room temperature, the deoxycholate tissue suspensions were centrifuged for 2 mm and the supernatants removed. The pellets were suspended in 1 ml of sa-

line, vigorously vortexed, and centrifuged for 2

mm. Following removal of the supernatant, the resulting pellet was again suspended in 1 ml of 4% deoxycholate, pelleted, twice suspended in imi of saline followed by centrifugation. The final pellet

was suspended in 0.1 N sodium hydroxide and heated at 60° C overnight to solubilize the basement

Isolation and incubation of rat kidney glomeruli. Male Sprague-Dawley rats weighing 250 to 300 g were used in all metabolic experiments. Glomeruli

membrane proteins for protein analysis. A 100-.tl aliquot was removed from each microfuge tube for

were isolated, as described previously [26], by a

Böhlen et al [29] with bovine serum albumin as a

procedure involving perfusion of the kidneys with a suspension of magnetic iron oxide and subsequent

isolation of the glomeruli containing trapped iron oxide with the use of nylon sieves and a magnet. The isolated glomeruli were incubated in Earle's balanced salt solution fortified with 50 mg/liter Pen-

protein assay by the fluorescamine method of standard. The remaining 900 l was neutralized with hydrochloric acid, taken up into scintillation cocktail, and counted. The scintillation cocktail consisted of two parts of a solution of 22.8 g of Omni-

fluor® (New England Nuclear, Boston, Mass.) per gallon of spectral grade toluene (Mathe son, Coleman and Bell, Norwood, Ohio), and one part Triton

icillin G® and Minimum Essential Medium Vitamins (Grand Island Biological Company, Grand Island, N.Y.) which contained per liter: 1.0 mg of Dcalcium pantothenate, 1.0 mg of choline chloride, 1.0mg of folic acid, 2.0mg of i-inositol, 1.0mg of nicotinamide, 1.0 mg of pyridoxal hydrochloride,

liliters of cocktail was necessary to form a monophasic system with 1.5 ml of aqueous sample. 14C-hydroxyproline determination. To measure the formation of [14C]-hydroxyproline from [14C]-

0.1 mg of riboflavin, and 1.0 of thiamine. When non-

proline, kidney glomeruli were incubated with [14C]-

radiolabeled amino acids were added to the incubation buffer, Minimum Essential Medium Amino Acid Solution (Grand Island Biological Company, Grand Island, N.Y.) was used. The re-

proline which had been purified by ion exchange chromatography. Purification of the [14C]-proline was necessary because of the presence of labeled material which cochromatographed with standard [3H]-hydroxyproline. After incubation, the deo-

sulting incubation amino acid concentration per liter was 105 mg of arginine hydrochloride, 24 mg of cystine, 292 mg of glutamine, 31 mg of histidine, 52.5 mg of isoleucine, 52.4 mg of leucine, 58 mg of lysine, 15 mg of methionine, 32 mg of phenylalanine, 48 mg of threonine, 10 mg of tryptophan, 36 mg of tyrosine, and 46 mg of valine. When radiolabeled amino acids were used in incubations, the corresponding amino acid(s) were omitted from the incubation medium. Incubations were carried out at

X-100 (Rohm-Haas Co., Philadelphia, Pa.). Ten mil-

xycholate soluble and insoluble materials were sep-

arately dialyzed against distilled water. The nondialyzable material was lyophilized, taken up into 6 N hydrochloric acid and hydrolyzed at 110° C for 18 hours. After hydrolysis, the hydrolysates were evaporated to dryness, taken up into 0.01 N hydrochloric acid, and placed on 200-mesh AG-50 x 8 ion exchange resin in the hydrogen form. The ion ex-

change resin columns were washed with distilled

NIche et a!

22

water followed by elution of the radiolabel by concentrated ammonium hydroxide. After evaporation, the residues were dissolved in 0.2 N sodium citrate

buffer (pH, 3.19). Separation of hydroxyproline from proline was effected by injecting a 250-il auquot of the citrate buffered sample onto a 40-cm by 0.5-cm Aminex-27 cationic exchange column (Bio Rad Laboratories, Richmond, Calif.), and by subsequent elution of the hydroxyproline and proline peaks from the column using the same buffer. Substrates and inhibitors. (N) [2, 3—3H]-L-proline (33.8 mCiJmmole), uniformly labeled ['4C]-L-

lucent areas. Capillary lumina occasionally contain fine flocculent materials (which may represent fixed

serum proteinaceous substances), but Bowman's (urinary) space—external to podocytes—is usually

free of noncellular material. Slit membranes frequently are present between podocytic foot processes. Following treatment with sodium deoxycholate, cellular elements of the renal glomeruli are solubilized, and only extracellular matrix (which is insoluble in this detergent) remains (Fig. 3). The preparation is virtually free of cellular debris and other

proline (232 mCi!mmole), uniformly labeled ['4C]-Llysine (312 mCilmmole), uniformly labeled [14C1-Lamino acid mixture, (G) [3HJ-L-amino acid mixture, [6— 3HJ-o-glucosamine (3.6 mCilmmole), and [3H]-

contaminants. Therefore, basement membranes and associated mesangial matrix are the primary

D-galactose (3.0 Cilmmole) were obtained from New England Nuclear Corp. [2-3Hj-Glycine (2.2 Cilmmole) was obtained from Amersham-Searle Corp. -Aminopropionitrile (BAPN), colchicine, and HEPES were products of Sigma Chemical

preservation of the glomerular histoarchitecture. Ghost-like profiles of capillary lumina, Bowman's

Company.

Phase-contrast and electron microscopy. Phase contrast photomicrographs of isolated rat renal glomeruli were obtained with the use of a Zeiss microscope equipped with phase optics. Fixation and electron microscopy of isolated glomeruli and gb-

merular basement membrane were carried out as described previously [261. Results

Morphology. Renal glomeruli isolated by out method are easily visible with a stereo dissection micro-

morphologic constituents. The most striking feature of these preparations, however, is the remarkable

space, and mesangial areas are easily recognizable.

Basement membranes remain intact and are not broken by the detergent procedure. At higher magnifications (Fig. 4), the glomerular

basement membrane appears as a homogeneous

sheet of finely filamentous material (2,000 A thick) which is ultrastructurally indistinguishable from its in situ counterpart. Mesangial matrix is present in these preparations and glomerular basement membrane is discontinuous beneath capillary endothehum in these regions. It is in all ultrastructural respects similar to that seen in situ. Basement membrane isolation and assay for incorporation of labeled precursors into extracellular Fig. 1. Phase contrast photomicrograph of isolated rat renal

scope. By this technique, we established that our

glomeruli (G). Glomeruli appear photodense because they have been perfused with colloidal iron (magnification, x428).

preparation is 99% pure and consists of a mixture of glomerular capillary tufts and glomeruli which re-

Fig. 2. Transmission electron micrograph of portion of rat renal

tained Bowman's capsule. At the level of phase-

glomerulus. Glomerular basement membrane (bm) separates capillary endothelium from visceral epithelium of Bowman's

contrast microscopy (Fig. 1) the corpuscles appear as darkly stained spheres. The heavy photodensity is a result of the accumulated iron oxide particles within glomerular capillaries following perfusion to facilitate their isolation. When individual glomeruli are fixed and conventionally prepared for transmission electron microscopy, they exhibit morphologic characteristics similar to those described in situ (Fig. 2). A thick (2,000 A) glomerubar basement membrane intervenes between fenestrated capillary endothelium and foot processes of podocytes. The basement membrane is flanked on either side by narrow (300 A) electron

capsule. CL is capillary lumen; BS is Bowman's space (x27,200).

Fig. 3. Low-power transmission electron micrograph of gbmerular basement membranes following treatment with sodium

deoxycholate. Glomerular histoarchitecture remains intact. Capillary lumena (CL) can be identified radiating from mesangial

(*) areas. These are clearly separated from Bowman's space (BS) by competent basement membranes (x4100).

Fig. 4. Transmission electron micrograph of area shown in rectangle in Fig. 3. A continuous, ultrastructurally intact basement membrane (bm) encloses the acellular capillary lumen (CL)

and separates it from the adjacent acellular Bowman's space (BS). Mesangial matrix (M) is identified at the base of the presumptive capillary lumen. Cellular membranes and other detergent soluble debris are not present in these preparations. Unit collagenous fibnls are virtually excludgd (x18,600).

23

Biosynthesis of gloneruIar basement membrane

CL

0

BS

*.

C

24

Hjel/e et al

Table 1. Effect of multiple saline washes in removing free [14C]-

amino acids from incubated rat glomerulia Wash no.

Saline soluble material cpm/ml saline 24,804 3,082 1,809

1

2

1233 137 103

Glomeruli from three rats were incubated as described in Methods with 20 Ci of 4C-amino acids. After 400 mm six 200-pi aliquots were removed, the glomeruli sedimented, and the medium discarded. The glomerular pellets were then resuspended and

washed three times with 1 ml of 0.9% saline, and the washes were counted (mean SEM).

matrix. To study de novo basement membrane formation, glomeruli isolated by the iron oxide perfusion method were incubated with radiolabeled amino acids and carbohydrates. By using deoxycholate to isolate the basement membrane matrix, cellular nonmatrix proteins labeled during the incubation are removed, but newly synthesized proteins deposited in the extracellular basement membrane matrix are retained. To determine the feasibility of such proce-

obtained in the third wash might be due to either residual extracellular material or leakage from intracellular sites.

To determine the effectiveness of deoxycholate extraction versus a combined sonication and deoxycholate treatment in completely removing label not incorporated into basement membrane matrix, we suspended each saline-washed glomerular sample in 1 ml of a 4% deoxycholate solution with vigorous vortexing. Half of the samples then were sonicated, whereas the other half were only vortexed.

After standing overnight at room temperature, the samples were centrifuged and the supernatants were removed and counted with the supernatants from two further deoxycholate washes. Table 2 de-

picts the loss of intracellular radioactivity from sonicated and nonsonicated deoxycholate-treated saline-washed glomeruli. An asymptotic decrease in the detergent-soluble radioactive material with each subsequent detergent extraction was observed. By the third wash, all detergent extractable counts had been removed. Sonication in deoxycholate was no more effective than was vigorous vortexing alone in

dures, we incubated glomeruli with a mixture of

removing nonmatrix incorporated counts. The

Ij'4C]-amino acids. After 150, 300, and 400 mm of

three-times-deoxycholate-treated glomeruli were subsequently washed with saline, resulting in the

incubation, multiple aliquots were removed, and the glomerular pellets were washed briefly several times with saline after removal of the incubation medium. Radiolabeled material in the combined sa-

line supernatants represented extracellular free amino acids. Table 1 shows the loss of radiolabeled material from extracellular portions of the glomeruii. The decrease in variability with increased number of washings suggested that the washing procedure could be performed in a consistent manner and that only small amounts of radiolabel remained after three washings. The small amount of radioactivity

extraction of only a minimal amount of radioactivity. Upon a final wash of the insoluble material with deoxycholate, no significant radioactivity was removed, indicating that any counts now remaining in the isolated purified basement membrane represented true incorporation into the extracellular matrix rather than trapping or binding. The increase in extracted counts with time reflected uptake and incorporation of radiolabel into glomerular protein. The remaining deoxycholate insoluble material was solubilized in 0.1 N sodium hydroxide and assayed

Table 2. Deoxycholate ex traction of radiolabel from isolated glomerul?

Deoxycholate solubie material Incubation time mm

150

Wash no. 1

2 3 300

1

2 400

3 1

2 3

Vortex only cpmlml deoxycholate 44,240

789

825± 146

100± 10

56,490 1,143

130±

79,723 2,394

1990

83 12

667 81

414± 68

Sonication and vortex cpm/ml deoxycholate 45,478

490

604± 10

47± 2

58,740 687 683

41

108± 20 79,700 1,599

886 90

329± 66

a Glomerular pellets from the experiment described in Table 1 were extracted with deoxycholate, as described in the text, and the extracts counted (mean of four samples saM).

25

Biosynthesis of glomerular basement membrane Table 3. Incorporation of radiolabel into isolated basement membrane after deoxycholate treatmenta Sonication and vortex

Vortex Incubation time

g protein

cpm

150

1953

300 400

2765 +

3671

51

125 107

126 4 117 7 116 8

15.49

23.88 32.22

g protein

cpm

Specific activity 0.27 0.65

2089

1.54

3089

1485

30 64 327

Specific activity

108 3

13.83 21.33 31.75

99 4 97

8

0.52 1.15 0.88

a The basement membrane pellets remaining after deoxycholate treatment in Table 2 were solubilized and counted (mean of eight

samples

SEM).

for radioactivity and protein as described in Methods. Table 3 shows the rate of incorporation of ['4C1-

amino acids into deoxycholate-insoluble extracellular matrix. Although the glomerular pellets which had been sonicated and vortexed in deoxycholate had a smaller number of counts, they also

had a correspondingly lower amount of protein, making the specific activity of the isolated basement

membrane obtained from these samples equal to that obtained by vortexing alone. With both treat-

ments, an increased incorporation of radiolabel with time was observed. Earlier experiments had shown that when large quantities of tissue were treated with deoxycholate,

a viscous gel, which was probably a complex of

25

DNA with basement membrane, was formed [1, 27,

20

15 U

x E

0 10

x E

5

0

40

160 80 120 200 Protein concentration, pg GBM

240

Fig. 5. Incorporation of [3H]-proline and [14C]-lysine into gbmerular basement membrane (GBM) as a function of protein concentration. Glomeruli from three rats were incubated as described in Methods with 100 Ci of [3H]-proline and 10 Ci of [14C]-lysine. After 330 mm, three aliquots each of 25, 50, 75, 100, 150, 200, 300, and 500 1.d were removed, and the amount of label

incorporated into basement membrane was determined. Closed circles (•——•) denote [3H]-proline, and open circles (0——C) denote [14C1-lysine.

240

0

300

360

Time, rn/n

Fig. 6. Incorporation of [3H]-glycine (•), l14C]-lysine (A), and [14C1-proline (•) into gbomerular basement membrane. Glomeruli from two or three rats were incubated as described in Methods with 67 Ci of [3H]-glycine, 10 Ci of [14C]-lysine or 10 Ci of [14C]-proline, three 200-1 aliquots were removed at each time point, and the amount of label incorporated into basement membrane was determined. The results with [14C1-proline consist of points obtained from three separate experiments.

26

Hje/le et a!

proline and lysine at basement membrane amounts of 20 to 200 g (Fig. 5).

Incorporation of radio/abe/ed amino acids and carbohydrates into basement membrane matrix. Using the assay described, we could demonstrate linear incorporation of radiolabeled glycine, lysine

and proline into the isolated, purified basement membrane matrix over a 5-hour period of incubation (Fig. 6). The time course of proline incor-

. a

0

200 Time, rn/n

. .

poration obtained in three separate experiments with different batches of glomeruli indicates the reproducibility of the incubation and assay system. A comparison of the radiolabel incorporated into iso-

lated basement membrane with that taken up and incorporated into deoxycholate-soluble proteins by glomeruli incubated with {'4C]-amino acids indicated that only 2.4% of the total counts taken up by the glomeruli are incorporated into basement

membrane (Fig. 7). Incorporation of the radiolabeled amino acids into both deoxycholate-soluble protein and insoluble basement membrane matrix was linear for over 6 hours.

Time, rn/n

Fig. 7. Incorporation of [C]-amino acids into deoxycholate soluble material and deoxycholate insoluble glomerular basement membrane matrix. Glomeruli from three rats were incubated as described in Methods with 20 sCi of ['4C1-amino acids, three 200-d aliquots were removed at each time point, and the amount of label in the deoxycholate soluble (inset) and insoluble fractions was determined.

£

'4-.

x 8

28]. This gel interfered with further extraction and manipulation of the tissue. To determine if a similar phenomenon might also interfere with glomerular basement membrane biosynthetic experiments, and

to establish that incorporation of radiolabel was proportional to the amount of glomeruli incubated, we determined the relationship between counts in-

corporated and quantity of basement membrane isolated, with {3H]-proline and ['4C]-lysine, two amino acids commonly used to study basement membrane synthesis (13—201. A linear relationship

between radiolabel incorporation and amount of glomerular basement membrane was seen for both

0

60

120

180

240

300

360

Time, rn/n

Fig. 8. Incorporation of [3H1-glucosamine (•) and 13H]-galac-

tose (A) into glomerular basement membrane. Glomeruli from three rats were incubated as described in Methods with 100 jCi of[3F1]-glucosamine or [3H]-galactose, three 200-st aliquots were removed at each time point, and the amount of label incorporated into basement membrane was determined.

27

Biosynthesis of glomerular basement membrane 10 -

Linear incorporation of carbohydrates into the extracellular basement membrane matrix was also demonstrated in the isolated rat glomerular system (Fig. 8). Glucosamine, a component of the heteropolysaccharide found in noncollagenous portions of the basement membrane [7-9], was incorporated readily into insoluble matrix, as was galactose, a component of dissacharide units of the collagenous portions of basement membranes as well as a component of heteropolysaccharide units [7-9]. Conversion of pro/me to hydroxyproline incorpo-

rated into basement membrane matrix. Since the collagenous moiety of glomerular basement membrane contains hydroxyproline, the synthesis and

deposition of basement membrane proteins into insoluble extracellular matrix can be followed by the appearance of radiolabeled hydroxyproline after incubation of glomeruli with labeled proline. Glomeruli were incubated with Ij'4C]-proline, and the appearance of ['4C]-hydroxyproline in the deoxycho-

late-soluble fraction and insoluble extracellular

0

8-

-Si

0 4-

0

'1

x E

20

60

120

180

240

300

360

Time, mm

Fig. 10. Effect of coichicine on incorporation of [3H1-amino acids

into glomerular basement membrane. Glomeruli from four rats were incubated as described in Methods with 50 Ci of [3H1amino acids in the presence and absence of l0 M colchicine, 0.5ml aliquots were removed at each time point, and the amount of label incorporated into basement membrane was determined. Closed circles (S——-•) denote control, and open circles (0——C) denote coichicine.

matrix was measured by hydrolysis of aliquots removed at different time points and separation of proline and hydroxyproline by ion exchange chromatography. The time course of hydroxylation, expressed as the percent of counts in hydroxyproline of the total proline counts, is shown in Fig. 9. The appearance of [14C]-hydroxyproline in the deoxy-

cholate-soluble fraction increases steadily in the first 4 hours of incubation and then plateaus at about 5% of the total proline counts. In contrast, the appearance of [14C]-hydroxyproline in the deoxycholate insoluble extracellular matrix is negligible during the first 4 hours and then increases to

x 0

0

close to 1% during the balance of the incubation. Inhibition of basement membrane synthesis. The incorporation of radiolabeled amino acids into basement membrane matrix was sensitive to inhibition by metabolic inhibitors such as sodium azide and

E

0 E

inhibitors of protein synthesis such as cycloheximide and puromycin, confirming that the activity observed was a true incorporation into base-

ment membrane proteins. Colchicine, an agent which inhibits collagen secretion, had no significant 4

6

8

10

Time, hours Fig. 9. Time course of [HCJhydroxyproline (HYDRO) appearance in the deoxycholate soluble fraction and deoxycholate insoluble glomerular basement membrane matrix. Glomeruli from four rats were incubated as described in Methods with 30 sCi of ['4C]-proline (PRO) 1-mi aliquots were removed at each time point, and the amount of label in proline and hydroxyproline in the deoxycholate soluble (0) and insoluble fractions (•) was de-

termined.

effect on the incorporation of radiolabeled amino acids into extracellular basement membrane (Fig. 10). f3-Aminopropionitrile (BAPN), an inhibitor of collagen crosslink formation, however, inhibited [3H]-proline incorporation into the glomerular base-

ment membrane, although the inhibition was not pronounced until after almost 6 hours of incubation (Table 4).

28

HjeIle et a!

Table 4. Effect of /3-amino propionitrite (BAPN) on proline incorporation into glomerular basement membranea (GBM)

Time mm

Control (dpmlmg x 10-s GBM)

BAPN

The procedure for basement membrane isolation which forms the basis for our assay of the biosynthesis of this extracellular matrix in isolated glomer-

uli yields material with a composition which is

Methods with 100 Ci of [3H]-proline in the presence and absence of 0.071 M BAPN; three 200-1.d aliquots were removed at each time point, and the amount of label incorporated into basement membrane was determined (mean 5EM).

chemically identical to that obtained by sonication techniques and which in contrast to the latter is unfragmented, histoarchitecturally intact, and remarkably free of contamination with cellular elements [1, 27—28]. The utility of detergents in the isolation of basement membranes and in monitoring basement membrane synthesis has been confirmed by other workers [34-38]. Although prolonged treatment of tissue subfractions with detergents could possibly result in proteolytic degradation of basement mem-

Discussion

brane components, the chemical composition of preparations obtained by such procedures is indistinguishable from those isolated with the use of

65 205

5.63 12.74

350

26.47

0.23

4.74

0.91 0.26

10.40 16.69

0.32 0.52 1.23

a Glomeruli from four rats were incubated as described in

The glomerular preparation used in these studies is greater than 99% pure with respect to freedom from tubular contamination and exhibits a wide variety of metabolic activities, including the oxidation of several substrates to carbon dioxide [26, 30], the synthesis of proteins and nucleic acids [301, and the

metabolism of kinins and angiotensins [31]. The presence of magnetic iron oxide in the capillary loops of these glomeruli has no effect on the oxidative metabolism of these organ subfractions [261, and this metabolic activity compared favorably with results obtained with kidney cortical slices [32], and with individual glomeruli, isolated by mild microdissection techniques, which are completely free of tubular contamination 1331. The magnetite particles trapped in the glomerular capillary loops are totally insoluble and wholly contained within the vessel lumens, and there is no evidence that they interfere in any way with the normal metabolism of the glomerulus [26, 311 although this possibility cannot be unequivocally excluded since rat glomerular preparations of comparable purity to those obtained with the use of magnetic iron oxide or tantalum are difficult, if not impossible, to obtain. Glomeruli isolated with the use of tantalum, a dense, chemically inert

metal similar to platinum, exhibited comparable metabolic activities [26, 31] to those containing magnetite, again confirming the metabolic integrity of the latter preparation. In addition, the morphologic characteristics of this glomerular preparation

isolated under conditions of mild perfusion with magnetic iron oxide are comparable to those seen in tissue sections [28] and are superior to those isolat-

sonication [1, 27—28, 36—37]; and in contrast to the latter, these preparations are ultrastructurally intact [1, 2, 27—28]. The inclusion of protease inhibitors

such as EDTA and phenylmethylsulfonylfluoride (PMSF) did not improve the chemical or ultrastructural integrity of the basement membrane prepara-

tions obtained by the deoxycholate procedure (Brendel and Meezan, unpublished observations).

It has been shown that bovine renal glomerular basement membranes isolated with sonication in the presence and absence of protease inhibitors yield virtually identical patterns of polypeptide components by SDS gel electrophoresis, suggesting that only minimal proteolysis occurs during isolation [39]. We have chosen in our initial studies of basement membrane biosynthesis in isolated glomeruli to fo-

cus our attention on the incorporation of radiolabeled precursors into the purified isolated extracellular matrix, since any amino acid and carbohy-

drate precursor of this structure may be used in such studies without concern for incorporation of the label into nonbasement membrane components. Thus, we are not limited to studying only biosyn-

thesis of collagenous portions of the basement membrane which would restrict our choice of labeled precursors to lysine and proline. Only by following the appearance of hydroxylysine and hydroxyproline into macromolecular components can the synthesis of soluble basement membrane precursors be studied [10, 16-18, 20], since no specific

membrane synthesis where it is important to ex-

assay for these components comparable to actual isolation of the basement membrane exists. Our assay combining detergent treatment of the glomerular pellets and extensive washing of the basement

clude all tubular metabolic activity.

membrane thus obtained, ensured that all counts as-

ed by microdissection [33]. They are, therefore, well suited to metabolic investigations on basement

Biosynthesis of glomerular basement membrane

29

sociated with this material were incorporated into the basement membrane matrix, as demonstrated by the linear relationship between counts of lysine and proline incorporated and amount of basement membrane isolated. This was confirmed by the inhibition of incorporation into isolated basement membrane by the metabolic poison sodium azide and the protein synthesis inhibitors cycloheximide and puromycin in agreement with studies where glomerular basement membrane was obtained by sonication

hydroxylation of proline in the deoxycholate soluble fraction, which would contain materials comparable to the soluble basement membrane precursors studied by other workers [16—18, 201, is five-fold greater than that observed in the insoluble basement membrane matrix. The appearance of hydroxyproline in the deoxycholate-soluble fraction was rapid within the first 2 hours and reached a plateau after 5 hours, in contrast to the appearance of this amino acid in the basement membrane ma-

[16, 19], as well as those in which incorporation into total glomerular protein was determined [14, 17, 18,

trix which was negligible until 5 hours and increased further thereafter. This presumably represented initial synthesis and secretion of basement membrane

401.

The total number of counts incorporated into the

collagen which was later deposited into the extra-

isolated basement membrane after incubation of glomeruli with a mixture of [14C]-labeled amino

cellular matrix. Although the ratio of ['4C1-hydroxy-

acids indicated that only a small fraction of the substrate taken up by the glomerular cells was utilized for basement membrane synthesis. This is in agree-

ment with evidence that the basement membrane has a very low turnover in mature tissues liii, 23— 251 and that basement membrane synthesis represents only a small fraction of the total protein synthetic activity of the glomerulus [13, 16, 17, 19, 201,

although the opposite viewpoint has also been expressed 11401. The same relationship between counts

taken up by the glomeruli and counts incorporated into deoxycholate-insoluble extracellular matrix was also true of the other radiolabeled compounds whose incorporation was examined. Incorporation of glucosamine and galactose into glomerular basement membranes has not been demonstrated previously, although the incorporation of these sugars into total glomerular protein has been reported [14, 151. Since glucosamine is a component

of the heteropolysaccharide found in noncollagenous portions of the basement membrane, the incorporation of this sugar demonstrates that noncollagenous glycoprotein components of this struc-

ture are being synthesized and deposited in the basement membrane matrix in this system. Incorporation of galactose into the basement membrane matrix indicated that the disaccharide moiety of the basement membrane was also synthesized by isolated glomeruli, although some galactose is known to be present in the heteropolysaccharide, and incor-

poration into this component may also be taking place.

Hydroxylation of proline and lysine has been used to monitor the biosynthesis of both soluble precursors and insoluble matrix components of the collagenous moiety of glomerular basement membranes [16-201. In our glomerular preparation, the

proline to [14C1-proline in the isolated basement membrane labeled in our experiments was less than

0.01, the ratio of these amino acids in unlabeled preparations of glomerular basement membrane is about 1. Similar lower than expected ratios of labeled hydroxyproline-to-proline and hydroxylysine-to-lysine have been reported in other studies

on the biosynthesis of the glomerular basement membrane [13, 17, 19, 201. There are several possible explanations for this discrepancy. The most likely is that synthesis of collagenous components

of basement membranes comprises only a small fraction of the total protein synthesized by the gb-

merulus in general and of that deposited in the basement membrane matrix in particular. This appears

to be the case in our system and in those reported by others [13, 16—181, although Beisswenger [19] found that more hydroxylation of lysine was observed in isolated basement membranes obtained by sonication than that found in the soluble fraction, the opposite of our results with proline. Another explanation for the lower hydroxylation levels seen by us may be damage to the hydroxylating system. The ratio of hydroxyproline-to-proline, however, increases in the deoxycholate-soluble fraction for the first 5 hours of incubation, indicating at least partial preservation of hydroxylation function. The

appearance of significant amounts of hydroxyproline in the basement membrane does not occur until 5 hours, indicating a lag in the secretion or deposition of collagenous proteins. A delay in the appearance of appreciable amounts of hydroxyproline in extracellular basement membrane components has been observed in both embryonic [41, 421 and mature [17, 18, 43] systems. This finding of a slow time course of appearance of hydroxyproline in the isolated basement membrane contrasts with the linear incorporation rates

30

Hjel/e et a!

droxyproline is presumably found only in the colla-

crosslinks are not important in the initial deposition of soluble precursors into the basement membrane matrix, but that lysine crosslinks might become a significant factor after several hours when they apparently enable certain components to be fixed into the matrix which would otherwise be extracted in

genous moiety, it is possible that noncollagenous

the deoxycholate soluble fraction. Evidence from

observed for glucosamine, galactose, lysine, proline, glycine, and the amino acid mixture. Since glu-

cosamine, galactose, lysine, proline, and glycine are all present in noncollagenous glycoprotein com-

ponents of the basement membrane, whereas hy-

proteins are synthesized and deposited into the basement membrane matrix at a faster rate than are the collagenous components of the basement mem-

brane. This might account for the lower than ex-

studies in glomerular basement membrane structure [9-11, 46] and biosynthesis [18, 47] indicates that disulfide bonds between glycoprotein and collagenous components of the basement membrane and

pected ratio of radiolabeled hydroxyproline-to-proline in the isolated basement membrane. From glucosamine and hydroxyproline incorporation experiments in the rat parietal yolk sac system, Clark et al [421 and Kefalides et al [44] proposed that basement membrane synthesis is a twocomponent system: a noncollagenous glycoprotein

between collagen chains themselves are of major importance in the organization of basement mem-

ference in the rate of radiolabeled [14C]-hydroxyproline appearance in the isolated basement membrane as compared to that of glucosamine, galac-

precursors into the extracellular matrix isolated

tose, lysine, proline, and glycine supports the

been used [12, 16, 19] or where total glomerular protein synthesis has been examined [13—18, 20, 40]. Although this investigation has focused on the bio-

and a collagenous protein. The significant dif-

concept of a two-component system for glomerular basement membrane synthesis as well. The relatively small amounts of collagenous basement membrane components deposited in the extracellular matrix in our experiments, and the possi-

bility of two or more components being synthesized, secreted, and crosslinked into the matrix at different rates was supported by our results with coichicine and BAPN. Colchicine, which disrupts the microtubular transport system involved in the secretion of procollagen from cells [45] had no significant effect on incorporation of [3H]-amino acids into the basement membrane matrix. Since colchicine only inhibits the secretion of procollagen and

not its synthesis or hydroxylation [45], it appears likely that in our glomerular system secretion of precursor collagenous components of the basement membrane matrix is not the rate-limiting step in the

synthesis of this structure. The fivefold excess of [14C]-hydroxyproline in the soluble fraction of the cells compared with that found in the isolated basement membrane is in agreement with the concept that secretion and synthesis of collagenous material by glomeruli is faster than its deposition into the ex-

tracellular matrix. This is also supported by the inhibition of [3H]-proline incorporation into the isolated glomerular basement membrane seen with the inhibitor of lysyl oxidase, BAPN. The inhibition observed with BAPN was not pronounced until after 5 hours of incubation, indicating that lysine-derived

brane structure. It is likely that the initial deposition of soluble components into the basement membrane matrix may involve the formation of such bonds. Our studies on basement membrane biosynthesis in purified preparations of isolated rat renal glomeruli indicate that the examination of incorporation of

with the use of deoxycholate can be a useful alter-

native to other procedures where sonication has

synthesis of components deposited into the extracellular matrix, the components in the deoxycholate-soluble fraction are also of interest in studying the processing of basement membrane procollagen and glycoproteins before they are laid down into the matrix. Studies on the collagenous precursors of the glomerular basement membrane [16-20] have yielded interesting results which correlate well with our findings on the isolated matrix.

Acknowledgments

Portions of this work were presented at the Federation of American Societies of Experimental Biology meeting in Los Angeles, 1976 (Fed Proc 35:680, 1976). This work was supported by USPHS Grants

Nos. AM

15394, AM 14977 and HL 17421-035 1, and

a grant from the American Heart Association. Dr.

of a Research Career Development Award from the NIAMD. We acknowledge Mr. Cliff Pollack for the phase contrast microscope picture of isolated glomeruli and Miss Betsy Hurd for assistance in the isolation of glomeruli. Meezan is a recipient

Reprint requests to Dr. Elias Meezan, Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona 85724, USA

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