Alterations in Glomerular. Proteoglycan. Metabolism in. Experimental. Non-Insulin. Dependent. Diabetes. Mellitusâ. Paola. Fioretto,. William. F. Keane,2. Bertram.
Alterations in Glomerular Experimental Non-Insulin Paola
Fioretto,
William
technical
assistance
P. Fioretto,
D.J. Klein,
F. Keane,2 of Frank
Proteoglycan Dependent
Bertram
X. Daniels
L. Kasiske, and
Greg
sity
of Minnesota
W.F.
Department
Medical
Keane,
School,
B.L. Kasiske,
G. Holden,
Department
M.P.
of Pediatrics,
Univer-
Minneapolis.
MN
O’Donnell,
of Medicine,
Medical
Center,
University
School,
Minneapolis,
MN
(J. Am.
Soc.
Nephrol.
1993;
of
microalbuminuria
before
the
develop-
ment of overt renal disease. The in vivo incorporation of (35S)sulfate into glomerular PG in 12-wk-old obese Zucker rats at the onset of microalbuminuria was compared with that of 12-wk-old lean Zucker rats. Specific
(35S)sulfate
incorporation
into
gbomerular
lean
rats.
Heparin
treatment
of isolated
uli released
an additional
HS-PG, which
be derived
primarily
the
I 2
Received August Correspondence
from
3. 1992. Accepted to Dr. W.F. Keane.
Nephrology, Hennepin neopolls, MN 55415.
County
Medical
gbomerubar
Center,
70 1 Park
1046-6673/03 10’-1694$03.00/0 Journal of the American Society of Nephrology Copyright C 1993 by the American Society of Nephrology
1694
gbomer-
appeared
to
extracellu-
November 23, 1992. Department of Medicine, Avenue
with
indicating
a hydrodynamic
size
similar
to
that
ropathy
in the
obese
Zucker
rat,
there
is increased
glomerular PG synthesis with no change in the proportions of the constitutively releasable and heparinreleasable HS-PG. Whether electrophoretic abnormalities of the heparin-releasable HS-PG observed in the obese rats contribute to the development of albuminuria and/or mesangial matrix expansion remains to be established. Key Words: rats,
Glomerular
non-insulin
glycans.
heparan
dependent
proteoglycans. diabetes
albuminuria. mellitus.
Zucker
glycosamino-
sulfate
PG
over 8 h was increased by 57% in obese rats compared with lean rats, suggesting increased PG synthesis. However, at variance with the observation in experimental models of insulin-dependent diabetes mellitus, the proportion of total gbomerular (35S)PG released by heparin treatment was unchanged. Heparan sulfate (HS)-PG constituted over 60% of radiolabeled de novo synthesized glomerular PG. Similar proportions of HS-PG were extracted from the glomeruli of obese and lean rats. Isolated gbomeruli spontaneously released two HS-PG, which constituted approximately 30% of total glomerular (35S)PG. On the basis of their chromatographic and electrophoretic patterns, these PG were similar in obese and
J. Klein,
for gbomerular basement membrane HS-PG. However, gel electrophoresis demonstrated faster migration of the HS-PG released by heparin from the gbomeruli of obese Zucker rats, suggesting increased electronegativity. Thus, early in the course of neph-
3:1694-1704)
occur
David
reported
Medical
ABSTRACT Glomerular proteoglycans (PG) are important in modulating extracellular matrix assembly and gbmerular permselectivity. In the obese Zucker rat, an experimental model of non-insulin dependent diabetes mellitus, expansion of the mesangial matrix, and
and
Holden
umns,
County
Minnesota
P. O’Donnell,
bar matrix compartment and not from the detergent soluble cell fraction. Heparin-releasable HS-PG from both the lean and obese Zucker rats eluted at a KAy of 0.31 from Sepharose CL-6B chromatographic col-
F.X. Daniels,
Hennepin
Michael
Metabolism in Diabetes Mellitus”
Division South,
of Mm-
T
he obese Zucker rat (OZR) is an autosomal recessive model of obesity and hyperbipidemia with many similarities to human non-insulin dependent diabetes meblitus (NIDDM) (1). Hyperinsulinemia and peripheral insulin resistance are evident in OZR at an early age (1). Fasting blood glucose is usually normal or slightly elevated during the first months of life, and modest fasting hyperglycemia is seen in older OZR (2.3). In OZR. small amounts of albumin are detectable in the urine at as early as 10 to 1 2 wk of age and this is accompanied by expansion of the mesangial matrix (2,3). We have shown increased mesangial type IV collagen. fibronectin. and laminin in OZR before the development of segmental gbomerubosclerosis (4). Recent experimental studies have demonstrated that abnormalities of lipid metabolism in OZR are important in the development of albuminuria, mesangial injury. and segmental gbmerulosclerosis (5.6). Glomerular proteoglycans (P0) regulate mesangial cell proliferation. modulate gbomerular extracellular matrix (GEM) assembly, and contribute to the permselectivity of the gbomerular filtration barrier (7-18). Reduction of gbomerular basement membrane (GBM)
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3
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1993
NIDDM
and
Proteoglycans
heparan sulfate (HS)-PG has been implicated as an early biochemical abnormality that may contribute to proteinuria in experimental models of diabetes (1 0- 1 5) and of the nephrotic syndrome ( 1 6, 1 7). In addition, studies in a murine model of NIDDM showed a relative reduction in renal cortical mRNA encoding for the core protein of the basement membrane HSPG (19). This reduction was correlated with the degree of albuminuria. Gbomeruli from streptozotocin-induced diabetic rats had decreased proportions of a heparin-releasable HS-PG (1 2). Although heparin treatment releases HS-PG associated with cell surfaces, the precise com-
partment(s),
cellular
or GEM,
from
which
heparin-
released PG were derived has not been established. We also evaluated whether differences in heparinreleasable components of gbomerular PG were present in OZR and bean Zucker rat (LZR) and determined whether these HS-PG were associated with the cell or GEM fraction.
MATERIALS Animal
AND
METHODS
Preparation
PG synthesis was studied in 1 2-wk-obd OZR and LZR (Charles River Breeders, Boston, MA). All animal experimentation described in this article was conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. In preliminary studies, we determined the relationship between iv dose of Na2[35S1O4 (International Chemical Nuclear Radiochemicabs, Irvine, CA). plasma I35SIsulfate level, and gbomerular incorporation of [35Slsulfate. Three to 4 days before the experiment. tail-cuff systolic blood pressures were determined in awake rats. Rats were then individually housed in metabolic cages for urine collection to determine albumin excretion. During the collection, rats were fasted but were allowed free access to water. After the urine collection, blood was obtained from rats under light ether anesthesia for the determination of plasma cholesterol, triglyceride. and glucose levels. As previously described (2-6). urine albumin was measured by nephebometry with a monospecific antibody to rat albumin. Plasma cholesterob, triglycerides. and glucose were measured coborimetricalby with an autoanalyzer as previously reported (2-6). Two days later, OZR and LZR received carrier-free Na2[35S104 iv. Rats were bled at 0.5, 1.0, 2.0, 4.0. and 8.0 h to measure serum I35SIsulfate concentrations. The rats were divided into groups of three animals each (four groups of OZR and four of LZR) and euthanized under ether anesthesia by exsanguination 8 h after the initial injection. Serum was separated for the determination of inorganic sulfate (20) and E35SIsulfate concentrations. Gbomeruli were isolated from decapsulated kidney cortices at 4#{176}C by serial sieving through stainless steel wire
Journal
of the American
Society
of Nephrology
meshes and were ments on the 100previously described
Gbomerular
(355)PG
retained with 95% of the total radioactivity) were used for the analysis of the heparan-[35S104 and chondroitin/dermatan-135S1O4 content by treatment with nitrous acid at bow pH or chondroitinase ABC (from Proteus vutgaris: Miles, Elkhart, IN). respectively. as previously described (9- 1 1). Ebectrophoresis in composite gels of 0.6% agarose and 1 .8% polyacrylamide was performed by methods modified from the work of McDevitt and Muir (23) as previously described (1 0- 1 2). Samples containing 1 ,500 to 2,000 cpm of I35Slsulfate from Sepharose CL-6B peaks were treated with chondroitinase ABC and heparitinase ( 1 01 2), ebectrophoresed at 20 mAmp/gel for 4 to 5 h, and stained with 0.2% toluidine blue in 0. 1 N acetic acid to visualize an Mr of 2.6 x 1 06 rat chondrosarcoma chondroitin sulfate PG standard and to fix the PG and GAG (10-12). The _
TABLE
1. Characteristics
of 12-wk-old Body
(g)
a b
OZR(N=12)
402±22b
LZR(N=12)
303±14
Abbreviations: p< 0.001.
BP, blood
pressure;
Wt
Zucker BP
(mm
Hg)
109±16 115± 12 UO,bV, urinary albumin
gels were impregnated for 1 h with En3Hance (Dupont, NEN Research Products) and exposed for 2 to 4 wks to x-ray film (Kodak. Rochester, NY) at -70#{176}C. The ratio of the distance from the origin to an autoradiographic band to the distance the PG standard migrated was designated as Rrc (1 0 12).
Histology Separate groups of 1 2-wk-obd OZR (N = 1 2) and LZR (N = 1 0) were used to examine gbomerubar morphobogy. Coronal kidney sections were fixed in Zenker’s solution, embedded in paraffin, and stained with periodic acid-Schiff. Mesangial matrix expansion and tububointerstitiab damage were assessed semiquantitatively with a 0 to 4+ scale, as previously described (2.5.6). The percentage of glomeruli with any evidence of gbomerubosclerosis was also determined. Gbomerubosclerosis was defined as areas of capillary collapse and replacement by periodic acid-Schiffpositive material (2,5,6). All tissue was examined in a blinded manner. Plasma obtained from these separate groups of rats was analyzed for sodium concentration with an ion-selective electrode with an autoanabyzer.
Statistical
Analysis
Data were expressed as mean ± standard deviation. Results between OZR and LZR were compared by t test. Data that were not normally distributed as determined by Kolmogorov-Smirnov analyses (e.g. . unnary albumin and serum triglycerides) were boganithmically transformed before the analyses. Regression analyses for the calculation of [35SJhabf-life were performed with the use of the Statistical Package for the Social Sciences (2-6).
RESULTS OZR weighed (Table 1 ). Blood OZR and LZR. creased in OZR. in OZR, whereas higher. compared
33% more than their lean littermates pressures were not different between Urinary albumin excretion was inTriglycerides were markedly elevated serum cholesterol was only 18% with values in LZR. Fasting blood
ratsa U0bV (mg/24 h)
4.13±4.2” 0.86±68
Cholesterol
Triglycerides (mg/dL)
73.4±8.9c 62.5±7.0
402.7±123.5k’ 58.0±6.9
Glucose
185.0± 118.2±
12b 11
Number
10
excretion.
Cp 0. 1). The area under concentration-time curve (AUC) of [35S significantly and directly correlated with I35Slmacromobecube incorporation (Figure
sulfate
there
distribution
I35Slsubfate molecules that the OZR.
was a larger in
OZR.
Despite
volume
of
lower
I5S1
plasma
in OZR, similar amounts of I35SImacrowere extracted from gbomerubi. suggesting synthesis of [35SIPG might be increased
in
80
Incorporation Macromolecules
S OZR I LZR
60
Because
[S]AUC
pCi/mI/hr 40
‘
I
.002
I
.003
I
.004
.005
.01
S] Sulfate Macromolecules (.tCi/mg protein) Gbomerular
[35
Figure 1 . Correlation between AUC for (35S)sulfate concentration-time-dependent levels and gbomerular (35S)macromolecules after iv administration of various amounts of Na2(35S)04 as described in Materials and Methods. The equation that describes this relationship is y = 4842.6x + 12.1, and the rvalue is 0.8 (P< 0.001). Each datum point represents the mean of three determinations from three rats. TABLE 2. Plasma
(35S)sulfate
levels
1#{189} (h)
OZR(N= LZR(N= 0
Groups
of
12) 12)
12 OZR
and 8 h to measure were serum
isolated, sulfate.
and
2.0± .41 1.95±29 and
12 LZP were
serum (‘S)sulfate (“S)sulfate
injected
and
gbomerular
incorporation
AUC/Inorganic Sulfate (MC/,zM per h)
57.95± 11.91b 70.40±8.13
51.37 ± 17.42b 79.18±12.79
with
(‘S)sulfate were
sulfate extracted
8 h before
being
for determination and
total
serum
of (355) in OZR and
Concentration-TimeDependent Serum (35S)Sulfate Levels (AUC; ,zCi/mL per h)
and inorganic
macromolecules
both
into
Gbomerular
inorganic
sulfate
and
I35Slsulfate levels were lower in OZR, we administered 30% more Na2[35SJ04 iv to OZR to assess PG synthesis rates at plasma [35Ssubfate levels comparabbe to those in LZR. After iv injection of 3.9 mCi of Na2[35SJO4 to OZR and 3.0 mCi to LZR. the t. for the disappearance of plasma [35SJ04 was similar in OZR and LZR (Table 2). Despite the 30% greater dose, plasma ISIO4 bevels were lower at all times in OZR and, thus, the concentration-time-dependent plasma I35SIsulfate bevels were significantly less (1 8%) in the OZR (Table 2). Inorganic sulfate was also lower in OZR than in LZR, 0.9 ± 0.04 versus 1 .2 ± 0.03 mM 504, respectively (P < 0.05). Thus, the SA of I35Slsubfate (AUC/inorganic sulfate) was 35% bower in OZR (Table 2). As shown above, the incorporation
20 C
of (35S)Sulfate
expressed
euthanized,
Gbomerular (35S)Sulfate Corrected for AUC/Inorganic Sulfate (x I 0)
Gbomerular (35S)Sulfate Macromolecules (zCi/mg of protein)
0.0113 ± 0.0108±001 and tail vein blood
of t and AUC as described as a function
LZR#{176}
of
glomerular
.001
2.2
.2”
±
1.4±08 samples
were taken
in Materials protein
at 0.5. 1. 2. 4.
and Methods.
content
and
Glomeruli
corrected
for
bp95% of the 135S1 macromolecules, was increased in OZR. Isolated glomeruli were incubated in media with or without hepanin to allow an evaluation of their constitutively released and hepanin-released PG contents, respectively. Cell-associated I35Slmacromolecules were then solubilized with a detergent-containing buffer. Electron microscopy of glomeruli treated with cell lysis buffer demonstrated that this treatment left the GBM and the mesangial matrix intact while removing the cellular components (Figure 2). The residual GEM fraction was then solubilized in 4 M guanidine-HCI-containing buffer. The proportions of [35Sjmacromobecules that were PG were calculated from the total radioactivity bound to DEAE columns after subtraction of the first peak (non-PG glycoproteins as determined by chemical and enzymatic analysis) and were comparable in OZR and LZR in each of the three fractions evaluated (Table 3). Approximately one third of gbomerular 135S] was spontaneously released into the media. whereas nearly one fourth was associated with the detergent-extracted fraction, representing cell-associated I35SIPG.
1698
TABLE ozRa
3.
Distribution
as described in Materials material (magnification.
of gbomerular
PG in LZR and
LZR
Heparin Medium
Treatment Fraction-%
Detergent-Extracted Total (35S)PG GEM Fraction-%
Total
(35S)PG
Fraction-% Total
(35S)PG
OZR
-
+
-
+
33
43
35
45
24
30
27
30
43
26
38
26
Groups of 12 OZI? and 12 LZR were injected with (35S)sulfate 8 h before being euthanized as described in Materials and Methods. Glomeruli were isolated and treated with RPMI 1640 with (+) or without (-) 2 mg/mL of added heparin for 30 mm at 37’C. The media and heparin-extracted glomeruli were then treated with 1% Triton X-100. 0.15 M NacI. 5 mM MgCI2. 2 mM EDTA, 0.25 mM dithiothreitol. I mM PMSF. and 10 mM Tris (pH 7.0) for 18 h at 4#{176}C (detergent extract fraction). Detergent-insoluble material was solubilized in 4 M guanidine HCI containing 0.05 M sodium acetate (pH 5.8) and protease inhibitors (see Methods. GEM fraction). The total (35S)PG content of each fraction was determined on the basis of retention by DEAE-Sephacei columns and sensitivity of its (‘5S)GAG to either enzymatic or chemical cleavage (see Methods). Materials insensitive to these treatments were designated (S)glycoproteins and were not included in the calculation of the percentages in each fraction.
0
The remainder of [35Slmacromolecubes was in the GEM fraction. Although hepanin has been shown to increase the release of [35S)PG from the gbomeruli (1 1 . 1 2). whether these PG were cell surface or extraceblubar matrix associated was unknown. In these experiments. the
Volume
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1993
,
..
.
.
...
.
.
,....
Fiorettoetal
L35S1
of constitutively released material from OZR gbomerubi is depicted in Figure 3A. The first peak (I) contamed [35Slglycoproteins. as determined by their lack of sensitivity to either nitrous acid or heparitinase and chondroitinase ABC treatment. The remaining two major peaks (II and III) contained HS-PG. No differences in the DEAE chromatographic profiles of constitutively released material were observed between OZR and LZR (data not shown). Constitutively released HS-PG contained in peak III ebuted with a
PG were purified initially by elution from DEAESephaceb columns. Three peaks were eluted at 0.15. 0.39, and 0.46 M NaCb concentrations. The DEAE chromatographic profiles of this constitutiveby released material were similar to those released by the addition of hepanin. A representative chromatograph
KAV of 0.3 1 when chromatographed on Sepharose CL-6B columns in both LZR and OZR (Figure 3B). These HS-PG migrated as two distinct autoradiographic bands on agarose-pobyacrybamide gel electrophoresis (Figure 4. lane 1 A and B). The Rrc of these two bands were 3.5 (A) and 4.3 (B). These were
increment in [35Sjmacromobecules released as a result of the addition of hepanin to the extraction media was accompanied by a similar decrease in the GEMassociated [35S]radioactivity. Thus, it appeared that hepanin-rebeasabbe [35SJPG were derived primarily from the GEM.
Spontaneous
and
Spontaneously
Heparin-Released
released
and
PG
heparin-released
,
A
8000
14 12
6000 0
10
U
U)
E
4000
E
8
V
‘6
U, 2000
C.,
4
Fraction
number
Fraction
number
3500 2000 3000 2500 C
2
2000.
U
1000
1500
E 0. V
1000
U) C,)
500
0 2
0.2
0.4
0.6
0.8
1.0
12
02
.
0
0.2
0 4
0 6
Figure 3. Purification of heparin-released and GEM HS-PG. OZR and LZR were euthanized 8 sulfate. Isolated glomeruli were treated with RPMI 1640 media with or without 2 mg/mL in Materials
and Methods.
with 4 M guanidine-HCL-containing after the removal of cellular
Cellular protease
of the
American
Society
of Nephrology
12
material inhibitors.
was solubilized Constitutively
after receiving an iv bolus of (35S) of heparin for 0.5 h at 37#{176}C as and the residual GEM was extracted
with detergent, released
(A) and
h
GEM-associated
components by detergent in the absence of heparin) (C) chromatographic columns with a salt gradient from 0. 1 to I .0 M NaCI in DEAE buffer. shown in panel A was seen after heparin treatment of glomeruli. Peaks of radioactivity constitutively released or heparin-released material were pooled in three separate constitutively released and the heparin-released associated HS-PG (indicated by a bar at similar positions (KAy. 0.3 1) from Sepharose CL-6B chromatographic columns run in the peak III from the GEM fraction (indicated by a bar in panel C), when run on a Sepharose the presence of detergent, had a KAy of 0.32 (D).
Journal
1 0
Kay
Kay
described
08
(guanidine
extracted
(35S)PG were eluted from HPLC-DEAE A chromatograph identical to that eluting at similar conductivities from fractions (peaks I through Ill). The under peak Ill in panel A) also eluted presence
of detergent
CL-6B chromatographic
(B). Similarly,
column
in
1699
NIDDM
and
Proteoglycans
1
2
34
Figure 4. Agarose-polyacrylamide spontaneously
released
gel electrophoresis of heparin-released gbomerular
and
(35S)PG. (35S)PG released
56
from gbomeruli
treated
with RPMI
media in the absence or presence of heparin were purified by HPLC-DEAE and Sepharose CL-6B chromatography. They were then electrophoresed in gels containing 1.8% acrylamide and 0.6% agarose either before or after treatment with chondroitinase ABC or heparitinase. Rat chondrosarcoma chondroitin sulfate PG was used as a marker (arrow). Two (355)PG were spontaneously released into the media in LZR (lane 1, A and B). They were HS-PG and completely resistant to chondroitinase treatment (lane 2, A and B). PG constitutively acteristics
released from (data not shown).
the OZR showed the same charHeparin treatment caused the
release of an additional heparan-(35S)-PG (HPLC-DEAE peak Ill, Figure 3A). In both LZR (lane 3) and OZR (lane 5), it migrated more slowly than did the spontaneously released PG in these gels. This PG was chondroitinase resistant (lanes 4 and 6) but entirely sensitive to nitrous acid deaminative cleavage, indicating that it was HS-PG in both LZR and OZR. The specific heparin-released HS-PG from OZR (lane 5) migrated faster towards the anode (Rrc 2.7 1 compared with ,
an R,c of 2.31
from
LZR, bane
3).
similar in LZR and OZR, and a representative electrophoretic gel from an LZR is displayed in Figure 4, bane 1 A and B. These I35SIPG were resistant to chondroitinase ABC digestion before gel electrophoresis (Figure 4, bane 2, A and B). This material was sensitive to nitrous acid digestion as shown by the elution profile on Sephadex G50 column chromatography (data not shown), indicating that HS-PG were the predominant constitutively released PG. In both LZR and OZR. DEAE peak II HS-PG eluted from the Sepharose CL-6B columns with a KAy of 0.43 (data not shown). Although not displayed. this peak contamed two HS-PG that migrated similarly to the peak III HS-PG in polyacrylamide gels. (For comparison. see Figure 4. lane 1 A and B).
The addition of heparin to the extraction media increased the amount of [35S]PG released by nearly 30% (Table 3). DEAE chromatography again revealed three peaks, which eluted at 0. 1 5, 0.38, and 0.46 M NaC1 concentrations, and the chromatograph was similar to the constitutively released PG depicted in Figure 3A. As in the constitutiveby released PG. DEAE peaks II and III were predominantly HS-PG as determined by chemical or enzymatic sensitivities, whereas peak I contained [35Slgbycoproteins in both OZR and LZR. In both LZR and OZR, DEAE peak II HS-PG from heparin-treated gbomeruli eluted from Sepharose CL-6B columns with a KAy of 0.43 (data not shown); peak II contained two HS-PG that also migrated similarly during gel ebectrophoresis to the constitutively released HS-PG from peaks II and III in both LZR and OZR (data not shown). DEAE peak III from heparin-rebeased fractions contamed a HS-PG that also ebuted from Sepharose CL6B columns at a KAy of 0.31 in both OZR and LZR, similar to that seen in the constitutively released HSPG (Figure 3B), thus indicating similar hydrodynamic size. Thus. in OZR and LZR, the chromatographic profiles of constitutively released and heparin-released HS-PG were not different. However, on gel electrophoresis in both LZR (Figure 4, lane 3) and OZR (Figure 4, bane 5). this heparin-rebeasable PG migrated more slowly than did the constitutiveby released PG (Figure 4. lane 1 A and B). In both LZR and OZR, this PG was chondroitinase resistant (Figure 4, lanes 4 and 6, respectively) but was completely sensitive to heparitinase digestion (data not shown). Interestingly. these heparin-rebeased HS-PG migrated to different positions after gel electrophoresis in LZR (Rrc, 2.31; Figure4. bane3)andOZR(Rrc. 2.71; Figure 4, lane 5). This difference in gel migration occurred despite a lack of alteration in hydrodynamic properties (KAy of ‘-0.31). suggesting that the heparin-rebeasable HS-PG from OZR may have increased subfation because its migration towards the anode was considerably faster. Alternatively. a small difference in the sizes of these HS-PG, undetectable by chromatographic techniques. could also explain this difference in migration. .
.
.
1700
Cell-Associated
PG
Detergent-extracted L35Slmacromolecubes eluted from DEAE columns as two peaks at 0. 1 5 and 0.40 M NaCl (data not shown). The first peak contained I35Slgbycoproteins with small hydrodynamic sizes, as suggested by high KAy (0.46 to 0.68). The second and major peak was further resolved by Sepharose CL-6B columns into a small void volume peak. which, by enzymatic digestion and agarose-pobyacrybamide gel electrophoresis, was determined to be a large HS-PG. The second and major peak ebuted at a KAy of 0.5 and contained predominantly intracellular chondroitin
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1993
Fiorettoetal
sulfate/dermatan sulfate GAG similar scribed in previous reports (24). None macromolecules had chromatographic retic characteristics resembling those the heparin-rebeased or spontaneously (see above), and they were not different and LZR.
GEM
to those deof these [35SJ or ebectrophodescribed for released PG between OZR
PG
All [35Slmacromolecubes after detergent treatment HC1. The relative amounts were consistently higher
remaining in the gbomeruli were soluble in guanidine of [35SIPG in these extracts in the gbomeruli not sub-
jected to heparin treatment (Table 3). Guanidineextracted [35Sjmacromolecubes from media-treated glomerubi eluted from DEAE-Sephaceb columns as three peaks at 0. 1 5. 0.39, and 0.48 M NaC1 in DEAE buffer (Figure 3C). There were no differences in the chromatographic profiles between LZR and OZR. The first major peak contained [35Sjglycoproteins, whereas the second contained both a large HS-PG (25%) and a CS/DS-PG (75%) (data not shown). The third and predominant [35SJPG peak ebuted from the Sepharose CL-6B columns at a KAy of 0.32 (Figure 3D). with a minor portion eluting later (KAy. 0.54 to 0.60). This HS-PG had a hydrodynamic size similar to that observed for the constitutively released and heparin-released HS-PG. In gels. it migrated in both OZR and LZR to an R of 2.8 (Figure 5. lane 1). similar to the Rrc observed for heparin-released HSPG. This PG was resistant to chondroitinase ABC (Figure 5. lane 2) but sensitive to heparitinase digestion (Figure 5, lane 3) and nitrous acid treatment. Importantly. after heparin treatment of gbomerubi from OZR and LZR, the GEM fraction no longer contamed this peak III (data not shown). Thus, it appears that the heparin-rebeasabbe HS-PG was extracted from the detergent-insoluble GEM. a fraction that contained both mesangiab matrix and GBM.
Figure
5. Agarose-polyacrylamide gel electrophoresis of (35S)PG. (35S)PG were extracted from the GEM of isolated LZR and OZR glomeruli with 4 M guanidine
GEM-associated HCI in the
presence
of protease
inhibitors
after
treatment
with RPMI media in the absence of heparin and detergent. After they were purified by HPLC-DEAE and Sepharose CL6B chromatography (Figures 3C and D, respectively), GEM HS-PG was electrophoresed in 0.6% agarose-1.8% polyacrylamide gels before (lane 1) or after treatment with chondroitinase
ABC
(lane
2) or heparitinase
(lane
3). Like
the heparin-rebeased HS-PG, this material was sensitive to both nitrous acid deaminative cleavage (not shown) and heparitinase digestion (lane 3) and was resistant to chondroitinase ABC treatment (lane 2). Similar electrophoretic findings were seen in LZR and OZR.
DISCUSSION In this study. we showed that [35Slsulfate incorporation into gbomerubar macromolecules was increased in the OZR. This was demonstrated after correction for alterations in the total sulfate precursor pool in OZR. as has been previously emphasized (25). Both inorganic sulfate bevels and [35Slsulfate were bower in OZR. Therefore, calculation of gbomerular I35Slsulfate incorporation relative to the plasma sulfate pool demonstrated a 57% increase in [355J macromolecules in glomeruli from OZR. The vast majority of gbomerular I35Slmacromolecubes (>95%) were PG as determined by biochemical analyses. The proportions and the hydrodynamic sizes of spontaneously released gbomerubar PG were comparable in gbomeruli from LZR and OZR. In
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experimental insulin-dependent diabetes mellitus (IDDM). in contrast, PG from this compartment were significantly increased ( 1 2). Thus, increased gbomerular PG synthesis in OZR was not associated with either the preferential appearance of a particular PG fraction or alterations in PG size at this albuminuric stage of nephropathy. Heparin treatment caused the release of similar amounts of [35SJHS-PG from the gbomeruli of LZR and OZR. However, there appeared to be a qualitative difference on gel ebectrophoresis of heparin-released HS-PG from OZR that could possibby be explained by increased ebectronegativity of this component. Our results also suggested that the GEM compartment (GBM and mesangial matrix) was the primary site from which heparin released HS-PG
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Proteoglycans
rather than from cell surfaces as previously specubated (11). The incorporation of [35Sjsubfate into the gbomerubar macromolecules of streptozotocin-induced diabetic rats was reported to be markedly reduced when compared with that of control rats (10-12,22.23). However, as in the OZR, gbomerular incorporation was increased when corrections were made for serum I35Slsubfate SA (1 1). Consistent with increased PG synthesis in streptozotocin diabetic rats, a slightly faster gbomerular [35S]PG turnover rate was also reported (26). Gbomeruli from streptozotocin-induced diabetic rats also contained decreased proportions of a heparin-rebeasabbe HS-PG ( 1 1 ). It has been suggested that this alteration might be a result of a disturbed interaction between PG and other matrix macromolecules as a result of nonenzymatic glycation (27). A decreased GBM HS-PG has also been proposed to contribute to gbomerubar permeability defects that underlie albuminuria in IDDM (28,29). However, recent clinical studies of GBM HS-PG in microabbuminuric IDDM patients failed to demonstrate a decrease by morphologic techniques in these anionic sites when compared with normoalbuminuric patients (30). In contrast. only in patients with clinically overt proteinuria was a decrease in anionic sites observed. These clinical data question the primary robe of reduced GBM HS-PG in the pathogenesis of microabbuminuria in diabetes. Recent dextran sieving studies in microalbuminuric diabetic patients also suggested that the increased transgbomerubar passage of albumin could be explained by alterations in the size selectivity of the GBM (3 1 ). However. a role for diminished charge selectivity could not be definitively excluded. In OZR. a model of NIDDM, heparin-releasable HSPG was not reduced, and thus, this did not contribute to the development of alterations in gbomerubar permselectivity and a slight increase in albuminuria. However, it is possible that qualitative alterations in the OZR HS-PG obtained from the GEM compartment may be important. A modest difference on gel ebectrophoresis in the migration of the heparin-rebeasabbe HS-PG in OZR compared with that in LZR (Figure 4, lanes 3 and 5) suggests qualitative differences such as increased ebectronegativity. It is possible that an increased electrostatic binding of these anionic HS-PG to type IV collagen could occur and result in alteration of type IV collagen assembly in the GBM (32), possibly contributing to abnormalities of pore size in the GBM (31). In addition, the role that this qualitative alteration in HS-PG might play in the expansion of the OZR mesangial matrix is unknown. The factors responsible for increased PG synthesis in OZR are unknown. Increased gbomerular hemodynamic function in experimental IDDM (33) has been proposed to alter glomerular processing of ex-
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tracelbular matrix proteins. However, comparable glomerular hemodynamic changes have not been reported in the OZR (3). The influence of the diabetic milieu on GEM biology has been only recently evaluated. Mild hypergbycemia, hyperinsulinemia, and marked dysbipidemia characterize the OZR. A high-glucose medium has been shown to increase mRNA encoding for type IV collagen. fibronectin, and laminin, as well as to increase the production of all three matrix proteins by cultured gbomerular mesangiab cells independent of insulin bevels (34). Increased medium glucose levels, per se, did not influence mesangiab cell production of PG but did reduce the stimubatory effects of insubin-like growth factor on mesangiab cell PG production (35). The role that insulin plays in altered GEM metabolism is unclear, although some studies have suggested that insulin may increase PG synthesis (36). Hyperlipidemia is severe in OZR (1 ). In the young OZR of this study, cholesterol was modestly elevated and triglycerides were increased more than sixfold compared with respective values in LZR controls. Atherogenic lipids have been shown to increase PG and type IV collagen synthesis in vascular smooth muscle cells (37). Whether lipids influence mesangial cell synthesis of matrix proteins remains to be established. Finally. recent studies have demonstrated an increased number of macrophages in the gbomerubi of OZR (5). Whether the presence of gbomerular macrophages in OZR influences PG metabolism is unknown. In summary, in OZR, the in vwo synthesis of gbmerular PG was increased when slight microalbuminuria was present. Biochemically. at this early stage of nephropathy. only modest qualitative changes were found in HS-PG released from the extraceblubar matrix compartment. Further studies to define changes in HS-PG subfation and how these might be involved in the altered GBM permselectivity of the OZR are required. Nonetheless, our results contrast with studies in the insulinopenic streptozotocin diabetic rat model, in which a heparin-rebeasable HS-PG appears to decrease before the development of abbuminuria. Indeed, our results are more consistent with recent reports of IDDM patients in whom no significant decrease in GBM HS-PG could be detected by histochemicab techniques during the microalbuminuric stage of diabetic nephropathy (30). Similarly. the role that the changes in PG metabolism play in the expansion of the mesangial matrix in obese rats needs further evaluation. Specifically. whether a biochemical modification in HS-PG, as suggested in our experiments. could directly contribute to altered processing of mesangiab matrix components as webb as GEM assembly needs further evaluation. Although it has also been established that PG
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are involved in modulating mesangial cell proliferation (38,39), whether these changes in gbomerubar HS-PG modulate mesangiab cell hypertrophy or hyperplasia and contribute to mesangiab expansion and increased gbomerular size in OZR (2) is unknown.
1 5.
16.
ACKNOWLEDGMENTS This study was supported in part by grants from the Extramural Grant Program of Baxter Healthcare Corporation. the Juvenile Diabetes Foundation, and the National Institutes of Health (ROl DK39786). We greatly appreciate the assistance of Ellen Davis in the preparation of the manuscript.
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