795
Effect of Diltiazem on Glomerular Heparan
Sulfate and Albuminuria in Diabetic Rats
Garikiparthy N. Jyothirmayi and Alluru S. Reddi
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Calcium entry blockers, particularly diltiazem, have been shown to lower not only systemic blood pressure
but also improve proteinuria in non-insulin-dependent diabetic patients. The presence of proteinuria is
attributed to the loss of glomerular heparan sulfate, which confers a negative charge on the basement
membrane. In the present study, we evaluated the efficacy of diltiazem in lowering blood pressure and
proteinuria in diabetic rats and also examined the possibility that diltiazem prevents proteinuria through
glomerular preservation of heparan sulfate. Diabetes was induced in male Wistar rats by streptozotocin
(60 mg/kg). One group of diabetic rats was treated with diltiazem (25 mg/L) in drinking water for 20
weeks. Another group of diabetic rats and a group of nondiabetic rats were given tap water only. Systolic
blood pressure was measured at 4, 8,12, and 20 weeks. Urinary excretion of albumin was done at 4, 8,12,
16, and 20 weeks. At the end of 20 weeks, all rats were killed, kidneys were removed, and glomeruli were
isolated. Total glycosaminoglycan and heparan sulfate synthesis were determined by incubating glomeruli
in the presence of [-"S] sulfate. Diltiazem lowered blood pressure significantly in diabetic rats at 8,12, and
20 weeks. Diabetic glomeruli synthesized less total glycosaminoglycan and heparan sulfate than glomeruli
from normal rats. Characterization of heparan sulfate by ion-exchange chromatography showed that the
fraction eluted with 1 M NaCI was significantly lower and the fraction eluted with 1.25 M NaCl
significantly higher in diabetic than in normal rats. Diltiazem therapy returned not only glomerular
synthesis but also various fractions of heparan sulfate to normal. Urinary albumin excretion was
significantly higher in diabetic than in normal rats; diltiazem therapy significantly lowered albuminuria
in diabetic rats. The data suggest that diltiazem therapy prevents albuminuria through preservation of
glomerular heparan sulfate in diabetic rats. (Hypertension 1993^1:795-802)
KEY WORDS • diltiazem • calcium channel blockers • proteoglycans • diabetes mellitus,
experimental • heparan sulfate • proteinuria
T
he pathology of the kidney in diabetes mellitus is
characterized by thickening of the glomerular
and tubular basement membranes and accumulation of basement membrane-like material in the mesangium.1 The onset of these changes is believed to be
signaled by the appearance of microalbuminuria (urinary albumin excretion of 30-300 mg per day).2-4 If
untreated, this microalbuminuria may progress to macroalbuminuria and then to nephrotic syndrome. It appears likely that albuminuria of either degree indicates
the escape of albumin across the glomerular basement
membrane (GBM). This escape appears to be due to
the loss of negative electrostatic properties of the GBM.
One constituent of the membrane, heparan sulfatecontaining proteoglycan, can confer a negative charge
on the lamina rara interna and externa of this structural
element. It therefore seems probable that a decrease in
heparan sulfate content would result in altered electrostatic properties and thus produce proteinuria.5 Indeed,
it has been shown, for example, that removal of heparan
sulfate from the GBM of rats by means of heparinase
From the Department of Medicine, UMDNJ-New Jersey Medical School, Newark, NJ.
Address for reprints: A.S. Reddi, MD, PhD, Department of
Medicine, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103.
Received August 31, 1992; accepted in revised form January 13,
1993.
treatment resulted in increased permeability to 12SIalbumin.6 A more recent study by Van Den Born et al7
showed that administration of a monoclonal antibody
against GBM heparan sulfate induced an increase in
urinary albumin excretion in rats. In human diabetic
subjects, a decreased content of heparan sulfate proteoglycan has been found in the GBM.8 Other evidence,
consistent with this hypothesis, is that decreased synthesis of renal proteoglycans has been reported in diabetic
rats9-12 and in Engelbreth-Holm-Swarm tumor grown in
genetically diabetic mice.13
In a previous report, we demonstrated a significant
decrease in glomerular synthesis of heparan sulfate
associated with an increase in albuminuria in long-term
diabetic compared with normal rats.14 Further studies
have shown that the changes observed in these diabetic
rats were prevented by treatment with angiotensin
converting enzyme (ACE) inhibitors, captopril,15 or
enalapril.16 The beneficial effects of ACE inhibitors in
preventing proteinuria in diabetic rats and human subjects have been well established.17"23 However, the role
of calcium entry blockers in the prevention of proteinuria in diabetic rats or human subjects has received little
attention compared with ACE inhibitors. Calcium entry
blockers are being extensively used to treat systemic
hypertension24'23 and to prevent renal damage.26-27 The
objectives of this study, therefore, were 1) to evaluate
the efficacy of diltiazem (DZM) (a benzothiazepine
Hypertension
\
FIGURE 1. Line graph shows elation patterns
of 35S-labeled heparan sulfate (standard) and
testicular hyaluronidase-digested glomerular material from Sephadex G50 column before and
after nitrous acid degradation.
i
- *>
i
i
i
i
5
4
3
2
1
0
i
i
i
i
i
ii
/
—A— sample + Nitrous acid
•••••0— tissue sample
— D — standard
r.'.>*
17
16
15
14
13
12
to
O 11
10
x 9
Vol 21, No 6, Part 1 June 1993
i
796
12 14 16
18 20 22 24 26 28 30 32 34
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fraction number
derivative of a calcium entry blocker) in lowering blood
pressure in diabetic rats, 2) to study the effect of DZM
on glomerular synthesis of heparan sulfate in diabetic
rats, and 3) to study whether DZM prevents albuminuria by preserving glomerular synthesis of heparan
sulfate.
Methods
Systolic blood pressures were measured in conscious
rats at 4, 8,12, and 20 weeks by the tail-cuff method. At
4, 8, 12, 16, and 20 weeks, each rat was placed in a
metabolic cage for 24-hour urine collection, at which
time the body weight and food and water intake were
recorded. After the total volume was measured, the
urine was centrifuged and used for determinations of
albumin, heparan sulfate, Na + , and K+.
A total of 26 male Wistar rats (Charles River) weighing 70-90 g were used in the study. After an overnight
fast, diabetes was induced in 17 rats with a single
intraperitoneal injection of streptozotocin in 0.1 M
citrate buffer, pH 4.5, at a concentration of 60 mg/kg
body weight. The remaining nine rats received an
equivalent amount of buffer and served as normal
control rats. One week after induction of diabetes, eight
diabetic rats were given ad libitum as drinking fluid tap
water that contained DZM (25 mg/L); the remaining
nine diabetic and nine nondiabetic rats were given only
tap water. Water was changed every day in all groups of
rats. The daily consumption of DZM was 5.72±0.65 mg
(mean±SEM). The drug treatment was continued for
20 weeks. All groups of rats were fed Purina rodent
chow (5001) ad libitum with the following composition
by weight: protein (23.4%), fat (4.5%), fiber (5.8%),
sodium (0.4%), calcium (1%), phosphorus (0.61%),
potassium (1.1%) and with vitamin supplementation.
The energy provided was 17.9 kJ (4.25 kcal/g).
Determination of Glomerular
Gfycosaminogfycan Synthesis
At the time they were killed, each rat was anesthetized with a single intraperitoneal injection of pentobarbital (5 mg/100 g) and bled from the abdominal aorta.
The kidneys were then removed, weighed, and the
cortices separated from the medulla. Glomeruli were
isolated from the cortex by differential sieving through
150-, 250-, and 63-/xm meshes,28 using ice-cooled 0.02
M phosphate-buffered saline, pH 7.2. After centrifugation at lOQg, the glomeruli were washed twice with
Krebs-Ringer solution, and the washed pellet was uniformly suspended in the same solution. About 10,000
glomeruli were incubated in 2 mL Krebs-Ringer containing 0.15 mg/mL glutamine, 50 fxg/mL ascorbate, and
50 /iCi/mL [3iS]sulfate (specific activity, 788 mCi/mmol)
in an atmosphere of 95% O2 and 5% CO2. After
incubation for 4 hours at 37°C, protein synthesis was
terminated by the addition of 1 mM puromycin. The
contents and further washings of the medium were
Animals
TABLE 1.
Data for Normal, Diabetic, or Diltiazem-Treated Diabetic Rats at End of Experimental Period
Glucose
(mg/dL)
K+
(mEq/L)
Creatinine
(mg/dL)
Urinary Na +
(mEq/day)
Urinary K+
(mEq/day)
6.39±0.48f
11.09±0.78
10.26±0.87
N+H 2 O (n=9)
>610
Kidney wt
(g)
3.90+0.09*
147±13t
3.38±0.24
0.82±0.04
3.90±0.4Ot
D+H 2 O (n=9)
325±31
5.10+0.33
514+65
4.02±0.37
0.97±0.11
5.84±0.49§
D+DZM (n=8)
366±31
4.92+0.18
454±58
4.53±0.27
0.74±0.07
8.10±0.88
Group
Body wt.
(g)
N+H 2 O, normal rats given tap water to drink; D+H 2 O, diabetic rats given tap water to drink; D+DZM, diabetic rats treated with
diltiazem. Values are mean±SEM.
"p<0.01, N+HjO vs. D+H 2 O.
tp<0.001, N+H 2 O vs. D+H 2 O.
+p<0.02, N+H 2 O vs. D+H 2 O.
§p<0.05, D+H 2 O vs. D+DZM.
Jyothirmayi and Reddi Diltiazem and Albuminuria in Diabetic Rats
797
200
D+DZM
M B D+HzO
E
TSSSA N+H 2 0
150
o
3
01
£
FIGURE 2. Bar gra/Vi shows systolic blood pressure in normal (N+H/D), diabetic (D+HjO), and
dMazem-treated diabetic (D+DZM) rats. Each
bar represents mean±SEM. "p< 0.5-0.007,
N+Hfi vs. D+Hfi; **p<0.05-0.001, D+Hfi
vs. D+DZM.
100
Q.
•o
O
_O
CD
50
20
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Weeks
transferred to a preweighed tube and centrifuged at
1,00% for 15 minutes. To the glomerular pellet was
added 2 mL acetone to remove lipids. This step was
repeated twice, and the pellet was dried and weighed.
The dried glomeruli were taken up in 2 mL of 0.1 M
acetate buffer containing 5 mM EDTA, 5 mM cysteine,
and 2.5 mI,/mT. crystalline papain (Sigma) and incubated at 60°C for 24 hours to release glycopeptides.29-30
Incubation was continued for another 24 hours with
addition of papain (1.5 mL/mL). At this time, 1 mg
carrier chondroitin sulfate (Sigma Chemical Co., St.
Louis, Mo.) was added. The glomerular glycosaminoglycans (GAGs) were precipitated with 0.3 mL of 10%
cetylpyridinium chloride. The precipitate was washed
twice with NaCl-saturated 95% ethanol, then with 95%
ethanol, and dried. The GAGs were further purified by
removing proteins and nucleic acids by precipitation
twice with 10% trichloroacetic acid and supernatants
combined. The pH of the supernatant was adjusted to 5
with 0.5 M sodium acetate, and GAGs were precipitated with 3 vol 95% ethanol. The precipitate was dried
and then dissolved in a known volume of distilled water.
An aliquot of this sample was used for quantitation of
radioactivity, which represented counts in total GAGs.
Total GAG synthesis is expressed as disintegrations per
minute per milligram glomerular weight or per
glomerulus.
To identify individual GAGs, another aliquot was
treated with 5 mg testicular hyaluronidase (Sigma) in
0.15 M NaCl and 0.1 M acetate buffer, pH 5.6, at 37°C
for 24 hours to degrade chondroitin sulfate. The digested material was placed on a Sephadex G50 column
(5x550 mm) and eluted with 0.15 M NaCl in 10%
ethanol. The included volume contained over 95% of
the uronic acid due to the degraded carrier, and the
radioactivity associated with this fraction represented
the counts in chondroitin sulfate. The voided volume
contained mostly heparan sulfate, and the radioactivity
associated with this fraction represented counts in
heparan sulfate. The presence of heparan sulfate in the
void volume was verified by its resistance to testicular
hyaluronidase and its degradation by nitrous acid (Figure 1).
Characterization of Heparan Sulfate
Since heparan sulfate is heterogeneous, it was further
fractionated on a Bio-Rad Agl-x2 (CT) ion-exchange
column (4x1 cm) to determine the distribution of
radioactivity within each fraction.31 The column was
eluted in a stepwise manner with 8 mL each of 1, 1.25,
and 2 M NaCl. Each fraction was dialyzed, dried, and
dissolved in distilled water; radioactivity was determined in each fraction. Since chondroitin sulfate was
removed with the testicular hyaluronidase and by elu-
TABLE 2. Characterization of Glycosaminogrycan Proteoglycaiu in Normal, Diabetic, and Diltiazem-Treated
Diabetic Rats
Glomerular wt.
Group
N+H2O (TJ=9)
D+HJO
(n=9)
D+DZM (n=8)
55.8±4.0
55.0±5.5
67.5±53
Incorporation of ["SJsuIfate into
total GAG
dpm/mg glom. wt.
dpm/glomerulus
251±37*
152±20
199±33
1.46±0.25t
0.77±0.16
1.32±0.26
Incorporation of
pSJsulfate into
heparan sulfate
Incorporation of
[35S]sulfate into
chondroitin
sulfate (%)
83.59±2.61'
66.91 ±6.96
85.79±3.76
16.38±2.61*
33.09±6.98
14.21 ±3.76
GAG, grycosaminogrycan; N+H2O, normal rats given tap water to drink; D+H2O, diabetic rats given tap water to
drink; D+DZM, diabetic rats treated with diltiazem. Values are mean±SEM.
•p<0.05, N+H2O vs. D+HjO.
1p<0.0l, N+HjO vs. D+HjO.
798
Hypertension Vol 21, No 6, Part 1 June 1993
TABLE 3. Percent Radioactivity in Glomeralar Heparan
Sulfate Fractions From Normal, Diabetic, or Diltiazem-Treated
Diabetic Rats
The gels were stained with Coomassie blue. The total
time required for separation and staining was 75
minutes.
Nad
Group
N+HjO (n=9)
D+HjO (n=9)
D+DZM (n=8)
1 M
1.25 M
2M
36.82±2.77*
31.97±0.17
35.26±1.34
29.12±2.19*
35.36±1.4O
32.05 ±1.38
34.05 ±1.83
30.98±131
33.91±1.71
Determination of Glucose, Creatinine, Na+, and K+
Plasma glucose was determined by the glucose oxidase method, using the reagents supplied by Sigma and
plasma creatinine by alkaline picrate reagent (Sigma).
Plasma K+ and urine Na+ and K+ were determined by
flame photometry.
N+H2O, normal rats given tap water to drink; D+H2O, diabetic
rats given tap water to drink; D+DZM, diabetic rats treated with
diltiazem. Values are mean±SEM.
Statistical Analysis
Data were analyzed by both parametric (Tukey*s test)
using one-way analysis of variance and nonparametric
method applying Kruskal-Wallis test and are expressed
as mean±SEM. The significance between two unpaired
means was evaluated by Student's t test and Fisher's
least significant detection test. A value of/J<0.05 was
considered significant.
V<0.05, N+H2O vs. D+H2O.
tion on Sephadex G50 columns, the radioactivity represented counts in heparan sulfate.
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Determination of Urinary Albumin
Urinary albumin was determined by the radioimmunoassay method of Brodows et al.32
Results
Table 1 shows pertinent information at the time the
rats were killed. Diabetic rats had significantly lower
body weight but higher kidney weight, protein intake,
plasma glucose levels, and urinary volume than normal
rats. DZM had no effect on any of these parameters in
diabetic rats. No differences in either plasma potassium
or creatinine were found among the normal, diabetic, or
DZM-treated diabetic rats. Urinary excretions of Na+
and K+ were higher in diabetic rats compared with
normal rats. DZM treatment significantly increased Na+
but not K+ excretion in diabetic rats.
Figure 2 shows systolic blood pressure levels at 4, 8,
12, and 20 weeks in various groups of rats. Blood
pressures were significantly higher in diabetic than in
normal rats. DZM lowered blood pressure significantly
in diabetic rats as shown at 8, 12, and 20 weeks of
treatment.
Table 2 shows the incorporation of [^SJsulfate into
total GAG and heparan and chondroitin sulfates by
isolated glomeruli in various groups of rats. A significant
decrease in incorporation into total GAG, when expressed either per milligram dry glomerular weight or
Urine Protein Electrophoresis
Randomly selected urine samples (20 weeks) from
one normal, one diabetic, and four diabetic rats treated
with DZM were used for electrophoresis, using
PhastSystem and PhastGel separation media (Pharmacia LKB Biotechnology, Uppsala, Sweden). Urine samples and low molecular weight standard protein samples
were prepared in 1 x sample buffer containing 10 mM
Tris/HCl, 1 mM EDTA, 2.5% sodium dodecyl sulfate
(SDS), and 5.0% B-mercaptoethanol at pH 8.0 and
heated at 100°C for 5 minutes. Approximately 0.01%
bromopheol blue was used as a tracking dye. Using an
8-track applicator, a 2 |tL sample containing 2 fig
protein per track was applied on a PhastGel homogenous medium (12.5% and 0.45 mm thick gel) with a
buffer system of 0.112 M acetate and 0.112 M Tris, pH
6.5. The gels were run with PhastGel buffer strips
containing 0.2 M tricine, 0.2 M Tris, and 0.55% SDS,
pH 8.1. (The buffer strips are made of agarose isoelectric focusing.) Optimal resolution of SDS-polyacrylamide gel electrophoresis (PAGE) was obtained at a field
strength of 250 V, 10.0 mA, 3.0 W, and 70 Vh at 15°C.
700
600 J5 500 j»
400
9
*300
O
m 200
- It
*
r
*
N+HjO
^ °
y
FIGURE 3. Line graph shows body weights in
normal (N+Hfi), diabetic (D+Hfi), and diltiazem-treated diabetic (D+DZM) rats. Each
point represents mean±SEM. *p<0.01-0.001,
N+Hfi vs. D+HjO; **p<0.05-0.025, D+Hfi
vs. D+DZM.
D+DZM
L...A-
D+H2O
100 12
16
Weeks
20
24
28
Jyothirmayi and Reddi Diltiazem and Alburainuria in Diabetic Rats
12
799
D+H20
t
CM
'"••••I""'
8
o
c
4
I
\
\ .I
J^
^rI .—
e
D+DZM
FIGURE 4. Line graph shows protein intake in
normal (N+Hfi), diabetic (D+Hfi), and diltiazem-treated diabetic (D+DZM) rats. Each
point represents mean±SEM. *p<0.02-0.001,
N+Hfi vs. D+Hfi.
N+HaO
*
*
"o
Q.
n
i
i
12
16
20
24
28
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Weeks
per glomerulus, was found in diabetic compared with
normal rats. Similarly, a decrease in heparan sulfate
synthesis was also observed in diabetic rats. However,
the incorporation into chondroitin sulfate was significantly increased in diabetic rats. Treatment of diabetic
rats with DZM returned these changes to normal. There
were no significant differences in glomerular weight
between the normal and the two groups of diabetic rats.
Percent radioactivity in various fractions of heparan
sulfate is shown in Table 3. The fraction eluted with 1 M
NaCl was significantly lower and the fraction eluted
with 1.25 M NaCl significantly higher in diabetic than in
normal rats. DZM corrected these fractions to normal.
No difference in the fraction eluted with 2 M NaCl was
found among various groups of rats.
Figures 3-5 demonstrate changes in body weight,
protein intake, and urine volume at various time intervals. As evident, both groups of diabetic rats had
significantly lower body weights than normal rats from 8
to 20 weeks of treatment (Figure 3). Protein intake
(Figure 4) and urine volumes (Figure 5) were signifi-
cantly higher in diabetic than in normal rats. Between
the two groups of diabetic rats, no difference either in
body weight, protein intake, or urine volume was
observed.
Twenty-four-hour urinary albumin excretion at various time intervals in all groups of rats is presented in
Figure 6. Diabetic rats had significantly higher urinary
albumin excretion than normal rats that increased with
duration of diabetes. DZM prevented this increase in
diabetic rats.
Figure 7 shows urine protein electrophoresis pattern
in normal, diabetic, and DZM-treated diabetic rats.
DZM-treated diabetic rats had lower concentrations of
albumin (molecular weight, 66 kd) when compared with
diabetic rats. Also, proteins corresponding to molecular
weights between 14 and 21 kd were prominent in
diabetic compared with normal rats.
Discussion
The present study demonstrates the following important findings: 1) DZM lowers systolic blood pressure in
FIGURE 5. Line graph shows 24-hour urine
output in normal (N+Hfi), diabetic (D+H2O),
and diltiazem-treated diabetic (D+DZM) rats.
Each point represents mean±SEM. *p<0.001,
N+Hfi vs. D+Hfi.
12
16
20
24
800
Hypertension Vol 21, No 6, Part 1 June 1993
FIGURE 6. Line graph shows 24-hour urinary
excretion of albumin in normal (N+Hfi),
diabetic (D+Hfl), and diltiazem-treated diabetic (D+DZM) rats. Each point represents
mean±SEM. *p<0.005, N+Hfi vs. D+H&.
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diabetic rats at a mean concentration of 5.72±0.65 mg
per day; 2) glomerular heparan sulfate and total GAG
synthesis are decreased in diabetic rats, and this decrease is prevented by DZM treatment; 3) urinary
excretion of albumin is increased in diabetic rats, and
this increase is prevented by DZM treatment; and 4)
DZM therapy does not affect either fasting plasma
glucose levels, body weight, kidney weight, plasma K + ,
or creatinine, but it promotes natriuresis in diabetic
rats.
Calcium entry blockers have been shown to have
variable effects on proteinuria in diabetic rats and
human subjects.26-27-33-34 Jackson et al35 demonstrated
lack of effect of verapamil (a phenylalkylamine deriva-
D + DZM
FIGURE 7. Pattern of urinary proteins on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis in normal (N),
diabetic (D), and diltiazem-treated diabetic (D+DZM) rats
for 20 weeks.
tive) on proteinuria in streptozotocin diabetic rats.
Mimran et al36 reported an increase in urinary albumin
excretion with nifedipine (a dihydropyridine derivative)
treatment in insulin-dependent diabetic patients with
incipient diabetic nephropathy (urinary albumin excretion >15 jig/min). This is in contrast to the results
reported by the Melbourne Diabetic Nephropathy
Study Group,37 in which nifedipine treatment significantly reduced albuminuria in hypertensive diabetic
patients. A decrease in exercise-induced microalbuminuria was observed with nifedipine in patients with
insulin and non-insulin-dependent diabetes.38 Holdaas
et al39 reported no effect of nifedipine on proteinuria in
insulin-dependent diabetic subjects. Nicardipine (a dihydropyridine derivative) has also been shown to reduce
albuminuria in non-insulin-dependent diabetic subjects.40 Bakris41 reported a significant reduction in proteinuria in diabetic patients with nephrotic syndrome
treated with DZM for 6 weeks. In a subsequent study,
DeMarie and Bakris42 compared the effects of DZM
and nifedipine on proteinuria in non-insulin-dependent
diabetic patients and reported a significant decrease
with the former and an increase with the latter drug.
Our results of a decrease in albuminuria in DZMtreated diabetic rats are consistent with the reports of
Bakris.41
The present study demonstrates not only a decrease
in the synthesis and concentration of heparan sulfate
proteoglycan in diabetic glomeruli, but also changes in
the type of heparan sulfate that is present. This is
evident from the elution pattern on ion-exchange columns with NaCl. These results are consistent with our
previous data in diabetic rats.14"16 It is known that the
sulfate content of the eluted fractions increases with
increasing salt concentrations, the heparan sulfate with
highest sulfate content being eluted with 2 M NaCl.43 A
significant decrease in the radioactivity of the 1 M
fraction in diabetic glomeruli indicates substantial loss
of low-sulfate-containing heparan sulfate compared
with normal glomeruli. On the other hand, a shift of
elution from 1 M fraction to the 1.25 M fraction may be
due to an increase in sulfation, a change in iduronic-
Jyothirmayi and Reddi Diltiazem and Albuminuria in Diabetic Rats
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glucuronic acid ratios, or an increase in molecular size
of heparan sulfate.44 This change in the diabetic glomerular heparan sulfate probably represents a different and
newly synthesized heparan sulfate. Since heparan sulfates are a heterogeneous group of anionic GAGs with
variable charge density, it is possible that diabetic
glomeruli synthesized heparan sulfate with different
physicochemical and functional properties. DZM therapy normalized glomerular changes of heparan sulfate
in diabetic rats. It is of interest to note that Nakamura
and Kojima45 reported that cancerous human liver
synthesizes a molecular species of heparan sulfate that
is lower in sulfate content than the heparan sulfate
synthesized by normal human liver tissue.
How DZM prevents glomerular and urinary loss of
heparan sulfate is not clearly understood. It is possible
that DZM may have lowered glomerular hypertension
early in the course of diabetes and thus protected the
glomerular capillary wall from thickening and leakage of
heparan sulfate. Recently, Anderson46 reported lowering
of glomerular hypertension by DZM in nondiabetic rats
with reduced renal mass. Additional data are needed to
evaluate pressure-induced biochemical changes in the
glomerulus and the basement membrane.
Increase in albuminuria with duration of diabetes in
untreated diabetic rats is not unexpected. However, it is
of interest to note that DZM therapy blunted this
increase without lowering plasma glucose. Reduction in
albuminuria was associated with glomerular preservation of heparan sulfate, suggesting an inverse relation
between charge density and albuminuria. The present
study emphasizes that DZM normalizes albuminuria
even in severely hyperglycemic rats. The improvement
in proteinuria is not related to changes in either dietary
protein content or urinary volume, since both untreated
and DZM-treated diabetic rats had similar food consumption and urinary volume. Furthermore, the observed decrease in albumin excretion in DZM-treated
diabetic rats was confirmed by demonstrating lower
concentrations of albumin on SDS-PAGE.
We have consistently observed an increase in chondroitin sulfate synthesis in diabetic rats. The significance of this increase is not clearly understood. Additional work is clearly warranted.
Acknowledgments
We thank Dr. Sheldon Gertner, Department of Pharmacology, for use of his equipment for blood pressure measurements
and Marie Birthwright for typing the manuscript.
References
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2. Viberti GC, Jarrett RJ, Mahmud U, Hill RD, Argyropoulos A,
Keen H: Microalbuminuria as a predictor of clinical nephropathy
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3. Parving H-H, Oxenboll B, Svendsen PA, Christiansen JS, Anderson AR: Early detection of patients at risk of developing diabetes
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4. Mogensen CE, Christensen CK: Predicting diabetic nephropathy
in insulin-dependent patients. N Engl J Med 1984^311:89-93
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the structural and functional basis of glomerular filtration and
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6. Rosenzweig LJ, Kanwar YS: Removal of sulfated (heparan sulfate) or nonsulfated (hyaluronic acid) glycosaminoglycans results
801
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Hypertension. 1993;21:795-802
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