113
Clinical Science (1990) 79,113-1 16
Glycosaminoglycan content, oxalate self-exchange and
protein phosphorylation in erythrocytes of patients with
‘idiopathic’ calcium oxalate nephrolithiasis
BRUNO BAGGIO, GIOVANNI MARZARO, GIOVANNI GAMBARO, FRANCESCO MARCHINI,
HIBBARD E. WILLIAMS* AND ARTURO BORSATTI
Institute of Internal Medicine, PostgraduateSchoolof Nephrology,University of Padova, Padova, Italy, and *Davis School of Medicine,
University of California, Davis, California, U.S.A.
(Received 4 September 1989/4 February 1990;accepted 16 March 1990)
SUMMARY
1. This study was performed to test the hypothesis that
glycosaminoglycans may play an important role in the
observed abnormalities in oxalate flux seen in patients
with calcium oxalate nephrolithiasis.
2. Oxalate flux rate, erythrocyte membrane glycosaminoglycan content, membrane protein phosphorylation
and effect of heparan sulphate on erythrocyte oxalate flux
in vitro were studied in control subjects and patients with
calcium oxalate nephrolithiasis.
3. In comparison with control subjects, renal stoneformers showed a significantly higher oxalate selfexchange, a lower erythrocyte membrane glycosaminoglycan content and a higher membrane phosphorylation rate. In stone-formers, erythrocyte glycosaminoglycan content correlated inversely with both
oxalate flux rate and protein phosphorylation. In vitro,
heparan sulphate promoted a sipficant fall in the rate of
oxalate self-exchange.
4. These findings support the hypothesis that a lower
erythrocyte membrane content of glycosaminoglycans
enhances membrane protein phosphorylation, leading to
an increased rate of transmembraneoxalateflux.
stilbene derivatives (such as 5,5’-dithiobis-2-nitrobenzoic
acid and 4,4‘-di-isothiocyano2,2‘-stilbenedisulphonic
acid) [3], which are known inhibitors of Band 3,
established that we were dealing with a true transport
mechanism and suggested a possible defect in the anion
carrier in ‘primary’ nephrolithiasis. In stone-forming
patients whose erythrocytes showed a high rate of oxalate
self-exchange, we also found an increased rate of
membrane protein phosphorylation [4], which, together
with the demonstration of a reduction in oxalatetransport
after depletion of erythrocyte adenosine 5’-triphosphate
[4], suggest that this anion carrier, like other ion
exchangers [5], requires phosphorylation to function
normally, raising the possibility of increased protein
kinase activity as a basis for this anomaly [6].
In looking for substances capable of interfering with
erythrocyte protein kinase activity, we focused on glycosaminoglycans (GAGS),since they have been reported to
inhibit casein, tyrosine and phospholipid-sensitive Ca2
dependent kinases [7-lo]. This study was set up to
challenge the hypothesis that a lower erythrocyte content
of GAGs could enhance protein kinase activity and Band
3 phosphorylation, leading to an increased rate of transmembrane oxalateflux.
+-
Key words: glycosaminoglycans, nephrolithiasis,oxalate.
Abbreviation: GAG, glycosaminoglycan.
INTRODUCTION
We have previously reported that the rate of oxalate selfexchange in the erythrocytes of renal stone-formers is
faster than normal [l, 21. The demonstration that this
exchange can be returned to normal by disulphonic
Correspondence: Dr Bruno Baggio, Istituto di Medicina
Intern, policlinico Universitario, via Giustiniani, 2, 35120
Padova, Italy.
METHODS
The study was camed out in 10 ‘idiopathic’ calcium
oxalate stone-formers (six males and four females, age
range 20-46 years), who had been selected in order to
obtain a widespread distribution of erythrocyte oxalate
self-exchange (from normal values to mean+ 10 SD),and
10 normal control subjects (seven males and three
females,age range 2 1-45 years).The diagnosisof primary
nephrolithiasiswas based on a normal standard urinalysis,
normal blood calcium, phosphorus, uric acid and parathyroid hormone levels, and a normal 24 h urinary excretion of adenosine 3’:5’-cyclic monophosphate. AU patients
114
B. Baggio et al.
had normal renal function as evaluated by creatinine
clearance. When the upper limit of 24 h urinary excretion
of oxalate was considered to be 0.50 mmol, that of
calcium 7.56 mmol and that of uric acid 4.96 mmol [ 111,
three patients were hyperoxaluric, two were hypercalciuric and one was hyperuricosuric. All control
subjects had a negative family history for nephrolithiasis,
normal routine urinalysis and normal renal function. In
both patients and control subjects, the erythrocyte oxalate
self-exchange, the GAG content and the erythrocyte
membrane protein phosphorylation rate were evaluated.
Oxalate self-exchange was also studied in erythrocytes of
patients with nephrolithiasis before and 1 h after addition
of heparan sulphate (Sigma)to the incubation medium at
a concentration of 5 mg/l.
Erythrocyte oxalate self-exchange was evaluated as
previously described [2]. Erythrocyte content of GAGs
and membrane protein endogenous phosphorylation were
determined in erythrocytic ghosts obtained as follows.
After platelets and leucocytes had been separated [ 121,
the cells were washed three times with isotonic phosphate
buffer (pH 8.0),lysed as described by Dodge et al. [13],
collected by centrifugation, washed three times with
buffer to remove haemoglobin and dialysed overnight
against 25 mmol/l Tris-HCI (pH 7.5). The GAG content
was assayed as described by Whiteman [14] in erythrocytes solubilized by 1% (w/v) Triton and was expressed in
pg/mg of protein determined by the method of Lowry et
al. [ 151. For endogenous phosphorylation, ghosts were
incubated at 37°C for 5 min in a medium containing 100
mmol/l Tris-HCI (pH 7.5), 8 mmol/l MgCI,, 2 pmol/l
adenosine [y3*P]triphosphate(about 10000 c.p.m./nmol)
and 90 pg of ghost proteins in a final volume of 112 pl.
The reaction was stopped by the addition of 14 pl of 20%
(w/v) sodium dodecyl sulphate and heating the mixture at
100°C for 5 min. An aliquot of the solubilized membranes
(approximately 50 pg) was submitted to electrophoresis
on a 0.1% (w/v) sodium dodecyl sulphate/lO% (w/v)
polyacrylamide slab gel as described by Laemmli [16].
The labelled proteins were quantitatively evaluated by
scintillation counting using a Packard Tri-Carb model 300
C counter, after staining and drying the gel. Phosphorylation values were expressed as c.p.m./mg of protein. Statistical analysis was carried out by the use of Student's t-test
for unpaired variables and of the coefficient of linear
correlation r.
RESULTS
Table 1 summarizes the oxalate flux rate, erythrocyte
membrane GAG content and protein phosphorylation
rate in patients and control subjects. In comparison with
control subjects, renal stone-formers showed a significantly higher oxalate self-exchange, a lower erythrocyte
GAG content and a higher phosphorylation rate. Furthermore, in stone-formers erythrocyte GAG content correlated inversely with both the transmembrane oxalate flux
rate (Fig. 1) and erythrocyte membrane protein phosphorylation rate (Fig. 2). Finally, the addition of a
constant amount of heparan sulphate to the incubation
medium of stone-former erythrocytes promoted a significant fall in the rate of oxalate self-exchange (3.02 f 1.59
vs 1.05f0.43x 10-2min-',mean+s~;t=4.79;P<0.001)
(Fig. 3).
DISCUSSION
We have previously shown an increase in oxalate selfexchange in the erythrocytes of idiopathic calcium oxalate
stone-formers [l, 21. This abnormality can be corrected
by Band 3 protein transport inhibitors [3] and is
associated with a higher phosphorylation rate of the
anion-channel protein [4], raising the possibility of a
defect in the anion-carrier phosphorylation as the basis
for the abnormality in oxalate transport.
Among the substances capable of interfering with
erythrocyte membrane protein phosphorylation, we
focused on GAGs, of which an inhibitory activity had
been reported on erythrocyte casein, tyrosine and Ca2+dependent phospholipid sensitive kinases [7-lo]. Furthermore, in the only study in which GAG synthesis by
fibroblasts from renal stone-formers was investigated, it
was found to be lower in stone-former cells than in
control cells [ 171.
The present study demonstrates that erythrocyte
membrane content of GAGs is reduced in patients with
renal stone disease, and that the transmembrane transport
rate of oxalate is indirectly correlated with their GAG
content (Fig. 1). Moreover, we also found an indirect
correlation between erythrocyte membrane GAG content
and membrane protein phosphorylation (Fig. 2). Finally,
the addition of GAGs to the incubation medium
normalized the abnormal oxalate self-exchange in
erythrocytes (Fig. 3).
Table 1. Oxalate self-exchange, erythrocyte GAG content and erythrocyte membrane protein
phosphorylation
Results are means k SD.
1 O2 x Oxalate
Control subjects
Patients
I
P
Erythrocyte membrane
self-exchange
GAG content
(min-1)
(pg/mg of protein)
Protein phosphorylation
(c.p.m./mg of protein)
0.30 f 0 . 1 3
3.05 f 1.63
5.32
<0.001
182.25 f 44.41
132.80 f 24.06
3.10
<0.01
64 265 f 4860
80 570 f 7105
5.98
< 0.001
Glycosaminoglycans,oxalate and nephrolithiasis
115
\
+/
100
140
180
GAG content (pg/mg of protein)
Fig. 1. Relationship between erythrocyte GAG content
and transmembrane oxalate flux rate. y = 10.21-0.05~;
r = -0.82. P<O.Ol.
Before
After
Fig. 3. Effect of heparan sulphate on erythrocyte oxalate
self-exchange in virro.
X
0
I
2
100
140
180
GAG content (pg/mg of protein)
Fig. 2. Relationship between erythrocyte GAG content
and erythrocyte protein phosphorylation. y = 112.520.24~;r = - 0.84; P< 0.01.
These observations,together with our previous demonstration that the addition of exogenous GAGs lowers
erythrocyte Band 3 phosphorylation in vitro [18],lead us
to propose the following hypothesis: a reduced GAG
content decreases the inhibition of protein kinases, which
enables a higher degree of Band 3 phosphorylation,
leading to a faster transmembrane oxalate transport. The
implication of this to oxalate transport by the gut and
kidneys are obvious and indicate the need for further
studies on the effect of GAGs on oxalate transport in
stone-formers. We have still to learn which kinase
controls Band 3 phosphorylation. If the behaviour of
heparin is paradigmatic for all GAGs, these substances
must be considered as rather aspecific protein kinase
inhibitors. In fact, heparin has been described as active
on at least three protein kinases, namely casein, tyrosine
and phospholipid-sensitive CaZ+-dependent kinases
[7-lo]. Although not investigated in this study, the possibility exists that the transport of other cellular ions
dependent on phosphorylation of the anion carrier are
also altered through the same mechanism. Indeed, the
increased erythrocyte Ca2+-Mgz+ adenosine triphosphatase found in hypercalciuric ‘idiopathic’ renal
stone-formers [19-20], together with the common observation not only of hyperoxaluria, but also of hypercalciuria and hyperuricuria in ‘idiopathic’calcium oxalate
renal stone disease,fit well with such a possibility.
The cause of the reduced concentration of GAGs in
erythrocyte membranes of renal stone-formers is not yet
known. Our study demonstrates a fall in erythrocyte
membrane GAGs, leaving unanswered the question of
whether the same thing occurs in the whole erythrocyte.It
is possible that in stone-formers, rather than a quantitative defect, there is a qualitativeabnormality which makes
GAGs bind less tightly to erythrocytes,so that their loss is
higher during membrane preparation.
A reduced urinary excretion of GAGs has been
reported in renal stone-formers, although inconstantly
[21-271. Owing to the inhibitory activity exerted by GAGs
on calcium oxalate crystal growth [28-321, their reduced
concentration in the urine has been considered a relevant
element for the pathogenesis of renal stone disease. This
study now raises the possibility that abnormalities in
GAGs may also effect oxalate transport in the erythro-
116
B. Baggio et al.
cytes of stone-formers which raises the possibility of a
widespread derangement in GAG metabolism in nephrolithiasis.
ACKNOWLEDGMENTS
This study has been partially supported by grants from
the Minister0 della Pubblica Istruzione and the Consigho
Nazionale delle Ricerche of Italy.
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