Diabetlgia (1985) 28: 928-932 Diabetlgia 9 Springer-Verlag 1985 Type IV cllagen antigens in serum f diabetic rats: a marker fr basement membrane cllagen bisynthesis D. G. Brcks 1, H. P. Neubauer I and H. Strecker 2 1Pharmaceutical Research Divisin, Department f Bichemistry and 2Radichemical Labratry, Hechst A. G. Frankfurt/Main, FRG Summary. The nature f type IV cllagen antigens in the serum f streptztcin diabetic rats was studied using radiimmunassays fr the N-terminal (7S-cllagen) and C-terminal dmain f type IV cllagen. Type IV cllagen antigen crssreacting with antibdies t the C-terminal dmain was elevated frm 32. 5.36 ng/ml (n= 1) in serum f nrmal rats t 94.9 + 24.5 ng/ml (n = 1, P<.1) in serum f streptztcin diabetic rats and culd be nrmalized t 4.1 8.3 ng/ml (n= 18) by insulin treatment. Mlecular sieve chrmatgraphy f serum demnstrated a high mlecular weight fractin cntaining the C-terminal and N-terminal dmains and small- er material cntaining nly the N-terminal dmain. Degradatin f the high mlecular weight material by cllagenase indicates that it cnsists f intact cllagen type IV. Its relative prprtin increased frm 42% t 54% 4 weeks after diabetes inductin. Tgether with unaltered clearance rates f 7S cllagen in nrmal and diabetic rats, the data suggest that the increase f cllagen type IV antigens in diabetic states reflects increased synthesis f cllagen type IV. Key wrds: Basement membrane, cllagen type IV, serum analysis, diabetes. Basement membrane thickening during the curse f diabetes mellitus is a well established fact [1]. In viv studies with radiactively labelled precursr amin acids have demnstrated increased glmerular basement membrane synthesis in diabetic rats [2-4]. Such experiments are, hwever, labrius and f limited usefulness fr fuwup studies. Recently radiimmunchemical methds have becme available t study basement membrane metablism by measuring serum cncentratins f basement membrane prteins like 7S-cllagen, the N-terminal crss-linking dmain f type IV cllagen, and laminin r laminin fragments [5]. arlier studies have shwn increased serum levels f 7S-cllagen in streptztcin diabetic rats which are nrmalized by insulin treatment [6]. It remains unknwn whether the increased serum levels are due t (a) a higher rate f synthesis f basement membrane cllagen; (b) different clearance rates f 7S-cllagen in diabetic and nrmal rats; r (c) an increased degradatin f basement membrane cllagen. In this study we reprt n experiments which favur the first interpretatin. Materials and methds Animals Nrmally-fed male Wistar rats f the strain H WISKF (Hechst AG, Frankfurt/Main, FRG), initially weighing 2-25 g, were used thrughut the study. Diabetes was induced by injectin f freshly disslved streptztcin (7mg/kg, Calbichem, Frankfurt/Main, FRG) int the tail vein. Rats treated with insulin were subcutaneusly injected with 9 IU Insulin (Lng Insulin, Hechst AG. Frankfurt/ Main, FRG) twice daily (1~ f the dse in the mrning, % f the dse in the late afternn). Methds The N-terminal and C-terminal dmains f type IV cllagen, 7S-cllagen and nn-cllagenus dmain t (NCt) were islated frm a transplantable murine tumur (HS), which prduced basement membrane material. The material was purified as described [7, 8]. Antisera were raised in rabbits by tw injectins f.5 mg antigen tgether with cmplete Freund's adjuvant at 4-week intervals. Antisera were cllected 4-8 weeks after the bster injectins. Serum levels f 7S-cllagen and f type IV cllagen NC1 were determined by radiimmunassay fllwing an established prtcl [9]. Radilabelling f the antigens with ~2sI was perfrmed by the chlramine T methd [1] fr 7S-cllagen and the Bltn and Hunter [11] methd fr NC1. The radiimmunassays were f the sequential saturatin type:.1 ml f antiserum at a dilutin capable f binding 5% f the tracer was incubated vernight at +4~ with.2 ml f unlabelled standard antigen slutin r an aliqut f the unknwn sample. Abut I ng f 1251- labelled antigen was then added US-cllagen: abut 2dpm in.1 ml, NCI abut 1 dpm in.1 rt), and the incubatin cntinued fr 6-7 h. Separatin f bund and unbund tracer was achieved by precipitatin with a secnd antibdy (gat antiserum t rabbit IgG -.5 U in.5 ml, Calbichem, Frankfurt/Main, FRG). Half-life studies Fr the evaluatin f the bilgical half-life f 7S-cllagen, 125I-labelled prtein (15 mci/mg) was injected int the tail vein f nrmal Wistar rats (H WISKF) (1 ~tci/15 g bdy weight, mean weight
D. G. Brcks et al.: Type IV cllagen anitigens in serum 929 1,,,,,,,,,,,,, \ 1" 8 6 4 ~! ' \ a 2 "i,5 lb 5 ng/ml l 5 2(~x),,All b 4 2 \\ ~ \~ ~* i 5 lb 5b ng/ml 31',25' 125 56 4.11 Fig. I a and b. Linearity f 7S-cllagen (a) and type IV cllagen NC1 (b) radiimmunassays. Standard curves (--) and serum inhibitin curves (- - -) f different rats are shwn.s 9 12 r- 28.t:: 4 _> el 4-6 8 1 12 14-16 age (weeks) Fig. 2. Serum cncentratin f type IV cllagen NC1 as a functin f age. The data are mean values _+ SD fr six rats in each grup, except fr the 2-, 4- and 6-week ld animals whse sera were pled ut f 6 individual sera Table 1. Recvery f type IV cllagen NC1 frm rat sera Amunt f NC1 (ng/ml) xperiment ndgenus Added Calculated Fund Yield a 48.2 7.5 55.7 53.5 96% b 48.2 3.9 79.1 77.5 98% 28 g) r streptztcin diabetic rats (1 #Ci/15 g bdy weight, mean weight 157 g) 4weeks after inductin f diabetes. At 2, 4, 8, 24 and 48 h intervals bld was taken frm the retrrbital venus plexus, and the radiactivity in serum was determined in a?'-cunter. In a secnd experiment a slutin f nn-labelled 7S-cllagen in PBS was injected int the tail vein f nrmal rats (9 ~tg/kg bdy weight, mean weight 3 g) and streptztcin diabetic rats (4 Ixg/kg bdy weight, mean weight 132 g) 7 weeks after diabetes inductin. At 1, 2, 4, 8 and 24 h intervals bld samples were taken and analyzed fr 7S-cllagen serum cncentratin by radiimmunassay. Backgrund levels f circulating 7S-cllagen amunted t 28.4 and 92.1 ng/ml in nrmal and diabetic rats respectively and were increased 1-fld by the treatment. Gel-filtratin chrmatgraphy Fr the determinatin f the size distributin f antigenic material present in serum, serum samples were analyzed by gel-filtratin chrmatgraphy, using Bigel A5m (2-4 mesh, 1.6 x 14 cm clumn) equilibrated in phsphatbuffered saline,.4% Tween2,.2% NAN3. Fractins f 2.2 ml were cllected. ach fractin was analyzed by radiimmunassay fr 7S-cllagen and NC1. The gel-filtratin clumn was calibrated using a standard mixture f glbular prteins (BiRad, Munich, FRG). Statistical analysis Statistical analysis was perfrmed using the ntailed Student's t-test fr unpaired data. Results Specificity f the radiimmunassays 7S-Cllagen. The sensitivity f the assay was 5.4.3 ng/ml (mean+ SD, n= 16) at the 5% intercept f the standard curve. The intraassay cefficient f variatin fr serum samples was 2.5% (n= 5), and the interassay cefficient f variatin was 6.% (n = 7). Recvery studies f exgenus 7S-cllagen added t serum revealed recvery rates between 84 and 112%. The linearity f the assay is shwn in Figure 1 a. Type IV cllagen NC1 The sensitivity f the assay was 5.8+.7ng/ml (mean 4- SD, n = 16) at the 5% intercept f the standard curve. The intraassay cefficient f variatin fr serum
93 D. G. Brcks et al.: Type IV cllagen anitigens in serum r._ ~-,,, 12-8- Z! T.1. samples was 2.7% (n = 5) and the interassay cefficient f variatin was 8.6% (n = 13). Table 1 shws the results f recvery experiments in serum. Satisfying recvery rates f 96 and 98% were fund. The linearity f the assay is dcumented in Figure 1 b. The age dependence f the cncentratin f NC1 antigen in rat sera is shwn n Figure 2. Highest values, which decreased gradually t reach stable values at an age f 1-12weeks (295-335 g bdy weight), were fund in infant rats..-= 4- _> c~ 4-1 N D D+I Fig.3. Type IV cllagen NC1 serum cncentratins in nrmal and streptztcin diabetic rats. Diabetes was induced by streptztcin 8 weeks prir t the study. Bld glucse cncentratin was 5.77 _+.72 mml/1, 22.2 _+ 5.5 mml/1 and 1.32 + 2.16 mml/1 in nrmal, diabetic and insulin treated diabetic rats respectively. Glycsuria was zer in cntrl grup (N), 6.6_ t.7 g/24 h in diabetic rats (D), and 3.1 _+ 1.3 g/24 h in diabetic+ insulin-treated rats (D + I). Mean bdy weight was 391 +34 g fr cntrls and 158 +24 g and 321 +25 g fr diabetic and diabetic + insulin rats respectively -~1~ { "5._<2 5 %.%_ l, 2 4 8 24 4'8 time(h) Fig.4. Disappearance f injected 125I-labelled 7S-cllagen frm rat serum as a functin f time. ----- = diabetic rats. H = nrmal rats. The mean value + SD fr 5 rats are given as a percentage f initial cncentratin measured 5 rain after injectin ~1- -~ ~~ 8~ ~6 lc 8g ' ' I l 2 4 8 2:4 time (h) Fig.& Disappearance f injected 7S-cllagen frm rat serum as a functin f time. ----- = diabetic rats; II----tl = nrmal rats. 7Scllagen cncentratin was measured by radiimmunassay after a single injectin f 7S-cllagen and given as mean fr 2 nrmal rats and as mean SD f 5 diabetic rats. Results are expressed as a percentage f initial cncentratin after injectin Type IV cllagen NC1 cncentratins in serum f diabetic rats Figure 3 shws that type IV cllagen NC1 is markedly elevated in serum f diabetic rats. This increase culd be nrmalized by treatment with insulin. Similar results have previusly been reprted when analyses were carried ut with a 7S-cllagen assay [6]. Half-life studies The increase in basement membrane antigens culd be due t a different clearance rate f these antigens in nrmal and diabetic animals. We therefre determined the bilgical half-life f radiactively labelled 7S-cllagen and f nn-labelled 7S-cllagen in bth grups f rats. Figure 4 shws the decline f radiactivity in the serum f nrmal and streptztcin diabetic rats after injectin f 125I-labelled 7S-cllagen. N difference in the bilgical half-life was fund. In rder t eliminate the pssibility f different levels f deidases being present in serum f nrmal and diabetic rats, a similar experiment was perfrmed with nn-labelled 7S-cllagen. The disappearence f antigen frm serum was fllwed by radiimmunassay (Fig. 5); it failed t shw any significant difference in the half-life between nrmal and diabetic rats. Nature f antigenic material in serum The nature f the material present in serum which shwed crssreactin in the radiimmunassays fr 7Scllagen and type IV cllagen NC1 has nt yet been determined. We analyzed the size distributin f antigenic material f serum samples frm nrmal and diabetic rats by gel-filtratin chrmatgraphy and radiimmunassays. Material pssessing 7S-cllagen epitpes emerged in tw peaks frm the clumn (Fig. 6 a), crrespnding in apparent mlecular weight t 7S-cllagen standard and ne with a cnsiderably higher mlecular weight. The relative amunt f 7S-cllagen in the latter peak was increased in sera frm diabetic rats (Fig. 6b). The relative amunt f antigenic material present in these tw peaks changed during the develpment f diabetes ver a perid f 4 weeks (Fig. 7). Analysis f the size distributin f antigenic material cntaining epitpes f NC1 demnstrated ne peak
D. G. Brcks et al.: Type IV cllagen anitigens in serum 931 6. r- '~,,s 5. e" ~4' 3 - r-2' ~1" a 2 4 6 fractin t- ~ @ b 12' 1-8, 6, 4 2, ee, T'2 2 4 6 fractin Fig.6a and b. Size distributin f type IV cllagen antigens in rat sera after gel-filtratin. A 1.5 ml serum sample frm a nrmal (a) r diabetic rat (b) was analyzed by 7S-cllagen (---) and type IV cllagen NC1 (----) radiimmunassay after gel-filtratin chrmatgraphy (Bigel A 5 m, 1.6 x 14 cm, phsphate buffered saline ph 7.2.4% Tween). Arrw: lutin Psitin f standard 7S-cllagen P~ -d 6 5 ~. 4 ~ r 2~ 3._ 2 1. > k~ duratin f diabetes (weeks) Fig.7. ffect f diabetes n the relative amunt f type IV cllagen antigen present in the high mlecular weight fractin after gel filtratin (see Fig. 6). Diabetes was induced in 4 rats by intravenus injectin f 7 mg/kg streptztcin (---); 4 cntrl rats were treated with.9% NaC1 ((3------). Bld was withdrawn at weekly intervals and analyzed after gel-filtratin chrmatgraphy fr 7S-cllagen. The relative amunt f 7S-cllagen is given in percent (mean_+ SD). Values at 1 and 3 weeks (p <.5) and at 4 weeks (p <.5) differed significantly. 6- A.5 = ~2- ".~ ~8" 6-._~ 4 2'._-_~~ ~ c.~. ~.g',-.~.,=~,_. 2 4 6 8 fractin Fig.8. ffect f cllagenase treatment n the size distributin f antigenic material related t type IV cllagen in serum. 1 ml f serum frm a streptztcin diabetic rat (NC1 cntent 11 ng/ml) was analyzed by gel-filtratin (------). Anther aliqut (1.5 ml) was incubated with.75 mg cllagenase (CLSPA Wrthingtn) fr 24h at rm temperature. 5 mml/1 DTA were added t stp enzymatic activity and 1.2 ml f the sample was analyzed by gel-filtratin (... ). The upper half represents 7S-cllagen radiimmunassay; the lwer half NC1 radiimmunassay 4 (Fig. 6). This peak cincided exactly with the higher mlecular weight peak detected by the radiimmunassay fr 7S-cllagen. The 7S and NC1 antigens were als analyzed in the serum frm a streptztcin diabetic rat by gel-filtratin chrmatgraphy befre and after treatment with bacterial cllagenase (Fig.8). The high mlecular weight peak cntaining NC1 material disappeared cmpletely after treatment and was cnverted t smaller material with apprximate mlecular weights f 4 kd and 2 kd. A hetergenus pattern f lw mlecular weight cmpnents was als detected in the treated serum by the radiimmunassy fr 7S-cllagen. When an albumin slutin was treated with cllagenase under similar cnditins n degradatin was seen (data nt shwn). Discussin The antigens studied in this paper - 7S-cllagen and type IV cllagen NC1 - frm the N-terminal and C-terminal dmains f type IV cllagen [12], the majr structural cmpnent f basement membranes [13-15]. arlier studies have shwn that 7S-cllagen can be detected in serum by radiimmunlgical methds [5]. The s
932 D. G. Brcks et al.: Type IV cllagen anitigens in serum rum cncentratin f this antigen is elevated in diabetic animals and can be nrmalized by insulin treatment [6]. The precise nature and metablic fate f the antigen measured in serum remains t be established. The cmparisn f clearance rates f 7S-cllagen in nrmal and diabetic animals (Figs. 4, 5) shwed a similar biphasic prfile. In the initial phase abut 8% f the antigen disappeared within 2-4 h, presumably due t tissue uptake f 7S-cllagen. The slwer phase we attribute t the degradatin and excretin f the circulating antigen. As n difference culd be detected between nrmal and diabetic rats, these experiments indicate that differences in the excretin rate cannt explain the elevated serum cncentratins in diabetic animals. Recently a methd became available fr the islatin f the C-terminal nn cllagenus dmain NCI f type IV cllagen [8]. Using a radiimmunassay specific fr this dmain, elevated serum levels were detected in streptztcin diabetic rats. These culd again be nrmalized by insulin treatment (Fig. 3). By using a cmbinatin f radiimmunassays specific fr the 7S and NC1 dmains f type IV cllagen, we culd discver sme features f a high mlecularweight frm f serum antigens. Bth assays detected a high mlecular-weight antigen which was prbably intact cllagen type IV r small ligmeric variants. As expected, this material culd be degraded by bacterial cllagenase, prducing NC1 antigens cmparable in size t its mnmeric and dimeric subunits [8] and a hetergenus ppulatin f small 7S cllagen peptides. The instability f the 7S epitpes indicates that the cllagen IV serum antigen is belw the size f tetramers which cause a stabilizatin against cllagenase [7, 12]. The analysis f nn-treated serum revealed a secnd antigenic peak which cntained 7S but nt NC1 epitpes (Fig. 6). This material was similar in size t authentic, tetrameric 7S cllagen [7] and presumably riginates frm tissue frms f crss-linked cllagen type IV. The type IV cllagen antigen in the high mlecular weight peak increases during the develpment f streptztcin diabetes. This indicates increased bisynthesis f type IV cllagen, and is in agreement with findings f Hasslacher et al. [16], wh shwed a psitive crrelatin between the rate f glmerular basement membrane synthesis and 7S-cllagen in serum. The data als shw that 7S-cllagen material in the secnd peak f rat serum has a mlecular weight cmparable t 7S-cllagen islated frm intact tissues. This material culd be due t degradatin f basement membranes; thus the 7S-cllagen may measure bth nesynthesis and catablism f type IV cllagen. The NCI radiimmunassay, hwever, detects primarily intact type IV cllagen and may be a suitable methd fr the exclusive analysis f increased basement membrane prductin. Acknwledgements. We are grateful t Ms. C.Steinert and Mr. M.Quint fr expert technical assistance, t Ms. R. Lhfink fr the preparatin f the manuscript and t Dr. R. Timpl fr stimulating discussins and valuable suggestins and cmments. References 1. Williamsn JR and Kil C (1977) Current status f capillary basement membrane disease in diabetes mellitus. Diabetes 26:65-73 2. Brwnlee M, Spir RG (1979) Glmerular basement membrane metablism in the diabetic rat. In viv studies. Diabetes 28: 121-125 3. Chen MD, Surma ML, Wu V-Y (1982) In viv bisynthesis and turnver f glmernlar basement membrane in diabetic rats. Am J Physi1242: F 385-389 4. Hasslacher Ch, Kpischke HG, Biirldin, Gechter F, Reichenbacher R (1982) In viv studies n basement membrane synthesis in diabetic and nndiabetic rats. Res xp Med 181:245-251 5. Risteli J, Rhde H, Timpl R (1981) Sensitive radiimmunassays fr 7S cllagen and laminin: Applicatin t serum and tissue studies f basement membranes. Anal Bichem 113:372-378 6. 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Timpl R, Wiedemann H, Van Delden V, Furthmayr H, Kiihn K (1981) A netwrk mdel fr the rganizatin f type IV cllagen mlecules in basement membranes. ur J Bichem 12:23-211 13. Kefalides NA (1973) Structure and bichemistry f basement membranes. Intern Rev Cnnect Tissue Research 6:63-14 14. Brnstein P, Sage H (198) Structurally distinct cllagen types. Annu Rev Bichem 49:957-13 15. Timpl R, Martin GR (1982) Cmpnents f basement membranes in: Furthmayr H (ed) Immunchemistry f the extracellular matrix, Vl II. CRC Press, Bca Ratn pp 119-15 16. Hasslacher Ch, Reichenbacher R, Gechter F, Timpl R (1984) Glmerular basement membrane synthesis and serum cncentratin f type IV cllagen in streptztcin-diabetic rats. Diabetlgia 26:15-154 Received: 14 March 1985 and in revised frm: 2 August 1985 Dr. D. Brcks Hechst AG Pharma Frschung Bichemie H 825 D-623 Frankfurt/Main 8 FRG