(Investigative Ophthalmology and Visual Science. 2000;41:1482-1485.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Erythrocyte Sodium–Lithium Countertransport and Proliferative Diabetic Retinopathy
Jacqueline M. Lopes de Faria1,2,
Lilia A. Silveira1,
Marcelo Morgano3,
Elizabete J. Pavin4 and
José B. Lopes de Faria1
1 From the Laboratory of Renal Pathophysiology, Nephrology Unit, Department of Internal Medicine, Faculty of Medical Sciences, UNICAMP, Campinas, SP, Brazil; the
2 Department of Ophthalmology, Faculty of Medical Sciences, UNICAMP, Campinas, SP, Brazil; the
3 Institute of Food Technology, ITAL, Campinas, SP, Brazil; and the
4 Endocrinology Unit, Department of Internal Medicine, Faculty of Medical Sciences, UNICAMP, Campinas, SP, Brazil.
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Abstract
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PURPOSE. To investigate whether elevated erythrocyte
Na+/Li+ countertransport
(Na+/Li+ CT) activity is present in patients
with proliferative diabetic retinopathy (PDR).
METHODS. The rate of Na+/Li+ CT activity assayed in 21
patients with type 1 diabetes mellitus (DM) presenting PDR was compared
with 10 patients with nonproliferative retinopathy (NPDR) and with 11
patients with normal fundi. Twelve normal volunteers with no family
history of hypertension were used as a control group. The albumin
excretion rate was determined by nephelometry, and the glomerular
filtration rate was measured by the plasma clearance of eidetic acid
labeled with chromium-51.
RESULTS. Patients with PDR showed higher diastolic blood pressure levels
(mean ± SD) compared with those with NPDR or normal fundi
(95 ± 13 versus 90 ± 09 and 82 ± 19 mm Hg,
P = 0.02, respectively). The albumin excretion rate
was higher [geometric mean (range)], and the glomerular filtration
rate was lower (mean ± SD) in patients with PDR than in those
with NPDR or normal fundi [333 (2 to 5140) versus 32 (5.9 to 2200) and
6 (1.5 to 306) µg/min, P = 0.01, and 63 ±
33 versus 99 ± 37 and 93 ± 43 ml/min, P = 0.02, respectively]. The mean Na+/Li+ CT in
patients with PDR was significantly higher than in patients with NPDR
or normal fundi and control group (0.46 ± 0.20 versus 0.32 ± 0.12, 0.32 ± 11, and 0.21 ± 0.07 mM/L red blood cells
(RBC)/h, respectively, P = 0.0001). In a multiple
logistic regression analysis, with PDR as the dependent variable,
Na+/Li+ CT (odds ratio [OR]: 4.7, confidence
interval [CI]: 1.2–17.6, P = 0.02), diastolic
blood pressure (OR, 3.4; CI, 1.3 to 9.6; P =
0.018), and glomerular filtration rate (OR, 5.1; CI, 1.6–17.7;
P = 0.007) were the only variables that were
maintained in the equation, indicating that they were the main
determinants of PDR.
CONCLUSIONS. Patients with type 1 DM and proliferative retinopathy have elevated
erythrocyte Na+/Li+
CT.
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Introduction
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Sodium–lithium countertransport activity
(Na+/Li+ CT) in red blood
cells has been found to be abnormal in subjects with essential
hypertension.1
The role of
Na+/Li+ CT in cell
physiology is still not clear, although this transport bears
similarities to the physiological sodium–hydrogen
exchanger.2
The
Na+/H+ exchanger is a
membrane transporter that regulates intracellular pH, cellular volume,
and growth and bicarbonate reabsorption by the proximal tubules in the
kidney.2
In patients with type 1 diabetes mellitus (DM),
cross-sectional studies have demonstrated that
Na+/Li+ CT is associated
with micro or macroalbuminuria.3
4
5
This finding
was recently confirmed by a prospective study.6
Because
this abnormality of cell membrane transport is probably the best
reproducible cellular cation transport abnormality phenotype associated
with essential hypertension in white individuals,1
it has
been suggested that a genetic predisposition to essential hypertension
is an important factor in the susceptibility to diabetic renal
disease.3
4
5
To the best of our knowledge, no study has
investigated whether elevated
Na+/Li+ CT is present in
patients with proliferative diabetic retinopathy (PDR).
The aim of the present study was to compare the rate of erythrocyte
Na+/Li+ CT activity in
patients with type 1 DM presenting with PDR to that observed in
patients with nonproliferative retinopathy (NPDR) or normal fundi, and
to explore the interactions between this abnormality in cellular
transport and other risk factors for PDR.
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Methods
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Patients
We estimated that to detect a difference of 0.20 ± 0.18 mM/L
red blood cells (RBC)/h, which has been reported in the literature for
white diabetic patients with and without microvascular
complications,3
a sample size of 17 patients in the
studied group will have a 90% power to detect that difference with a
two-tailed significance level of 0.05.
From January 1997 to July 1998, consecutive patients with type 1 DM
attending the diabetic outpatient clinic at the University Hospital of
the State University of Campinas were asked to participate in the
study. The criteria for inclusion in the study were patients with type
1 DM, defined as age at diagnosis less than 35 years, a history of
sudden onset of severe hyperglycemic symptoms, marked weight loss,
spontaneous sustained ketosis or ketonuria, an age ranging from 18 to
45 years old, white, with DM for at least 10 years (to allow enough
time for developing of diabetic retinal disease) and free of any
endocrine, hepatic, metabolic, or cardiac disease, and nondiabetic
renal disease. The exclusion criteria were pregnancy and use of
contraceptives or estrogen. Such criteria were deemed appropriate to
exclude patients with factors that may influence the determination of
Na+/Li+ CT.7
Patients with high myopia, chorioretinitis scars, posterior uveitis,
and glaucoma were also excluded, as were those who had undergone a
previous ocular surgery, because such events may influence the
development of diabetic retinopathy.8
Of the potential 80
patients for the study attended at the outpatient clinic, 38 (48%) did
not meet the inclusion and exclusion criteria. The remaining 42
patients gave their informed consent before participating in the study.
Twelve normal volunteers with no family history of hypertension were
used as a control group. The study was carried out in conformity with
the tenants of the Declaration of Helsinki, which was approved by the
Ethics Committee of the University Hospital. All patients were treated
with insulin, and 17 (41%) who were taking antihypertensive drugs were
asked to discontinue them for 48 hours before the protocol.
Clinical and Ophthalmologic Measurements
The patients attended the outpatient clinic after an
overnight fast. Blood pressure (BP) was measured twice, in the supine
position, by a single observer (JMLF), to the nearest 2 mm Hg, using a
standard cuff, 5 minutes apart after the subject had been resting for
at least 5 minutes. Mean systolic and diastolic (fifth Korotkoff’s
sound) blood pressures were obtained by averaging the two measurements.
A blood venous sample was taken for the measurements of RBC
Na+/Li+ CT activity and
biochemical analysis. A complete ophthalmologic examination was then
performed, including indirect fundus ophthalmoscopy with stereoscopic
color fundus photographs of seven standard fields of both eyes. The
stereoscopic color photographs were examined by a single observer
(JMLF) unaware of the patient’s
Na+/Li+ CT status. The
level of DR was classified according to the Early Treatment
Diabetic Retinopathy Study retinopathy severity
classification.9
The eye with the most severe level of DR
in each subject was considered for analysis.10
Eyes
presenting level 1, levels 2 to 5, and levels 6 to 8 were grouped into
normal fundi, NPDR, and PDR, respectively.
Methodology
RBC Na+/Li+ CT
activity was measured according to the original method described by
Canessa et al.,1
as previously reported by our
laboratory.11
In our laboratory, the normal range for
normotensive subjects with negative familial history of hypertension
(n = 12) was 0.21 ± 0.07 mM/L RBC/h, ranging from
0.12 to 0.38 mM RBC/h.11
The intraindividual and
interassay variations were approximately 9.2%, a value similar to that
reported previously.
Other Measurements.
Three consecutive timed overnight urine samples per patient were
collected for albumin measurement. Albumin concentrations were
determined by nephelometry. The median value of the three specimens was
used for classifying the patients into three categories:
normoalbuminuric (albumin excretion rate [AER] 
20 µg/min),
microalbuminuric (20 < AER 200 µg/min), and
macroalbuminuric (AER > 200 µg/min). Glomerular filtration rate
(GFR) was measured by the plasma clearance of eidetic acid labeled with
chromium-51 (51CR-EDTA). For comparison between
patients, GFR was corrected for body surface area (1.73
m2). The percentage of the
A1c component of glycosylated hemoglobin was
assessed by high-performance liquid chromatography. Normal values in
our laboratory were 5.6% ± 1.3%. Serum creatinine, total
cholesterol, and triglycerides were measured by an automated method. In
our laboratory, the normal values for serum cholesterol and
triglycerides were up to 5.20 and 2.30 mM, respectively.
Statistical Analysis.
Clinical data and laboratory determinations were compared for
significance using ANOVA, Fisher and Scheffé F tests from a
commercial software package (SPSS for Power Macintosh). Because of the
interrelationship of several variables, the univariate logistic
analysis was followed by multiple logistic regression analysis, with
PDR as the dependent variable. The results were expressed as the
mean ± SD, unless otherwise stated. The null hypothesis was
rejected below the conventional (two-tailed) 0.05 level.
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Results
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Among the studied group, 21 patients had PDR, 10 patients
presented NPDR, and 11 patients had normal fundi. The demographic and
laboratory characteristics of the patients are summarized in
Table 1
. The three groups of patients had similar distributions for sex, age,
body mass index, duration of DM, and systolic BP. However, patients
with PDR had diastolic BPs significantly higher than those with NPDR
and normal fundi (95 ± 13 versus 90 ± 09 and 82 ± 19
mm Hg, P = 0.02, respectively) and were more likely to
be on antihypertensive therapy (P = 0.001; Table 1 ).
The metabolic control, evaluated by HbA1c levels,
was similar in all groups of studied patients (Table 1)
. AER (geometric
mean [range]) was higher, and GFR (mean ± SD) was lower in
patients with PDR than in those with NPDR and normal fundi (333
[2–5140] versus 32 [5.9–2200] and 6 [1.5 to 306] µg/min,
P = 0.01; and 63 ± 33 versus 99 ± 37 and
93 ± 43 ml/min, P = 0.02, respectively). The
frequency of patients with micro or macroalbuminuria was higher in
patients with PDR (80%) than in those with NPDR (40%) and normal
fundi (18%) PDR, P = 0.001. In addition, the mean
level of the total cholesterol was significantly higher in patients
with PDR than in the other groups (6.53 ± 1.80 versus 5.10 ± 0.80 and 4.8 ± 1.2 mM, P = 0.008).
Figure 1
shows the individual values of the erythrocyte
Na+/Li+ CT activity. The
mean value for Na+/Li+ CT
activity in patients presenting PDR was significantly higher than that
observed in patients with NPDR, normal fundi, or control group
(0.46 ± 0.20 versus 0.32 ± 0.12, 0.32 ± 11, and
0.21 ± 0.07 mM/L RBC/h, respectively, P ±
0.0001). This difference in
Na+/Li+ CT activity
remained significant even when the micro and macroalbuminuric patients
were excluded from the calculation (0.55 ± 0.29 versus 0.32 ± 0.09 and 0.34 ± 0.12 mM RBC/h, P < 0.05 for
patients with PDR, n = 4; NPDR, n = 6;
and normal fundi, n = 9; respectively), suggesting that
the difference in mean
Na+/Li+ CT activity cannot
be accounted for solely by this group of patients. The presence of
laser therapy had no effect on
Na+/Li+ CT because the
activity of this cation transport was similar in patients with PDR that
had laser therapy (n = 8) and those without laser
therapy (n = 13; 0.48 ± 0.21 versus 0.42 ±
0.20 mM/L RBC/h, P = 0.60). The
Na+/Li+ CT activity was
above the upper limit of normal (>0.38 mM RBC/h) in 12 of 21 patients
with PDR (58%), in 2 of 10 patients with NPDR (20%), and in 2 of 11
patients with normal fundi (18%; P = 0.03).
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Figure 1. Rates of RBC Na+/Li+ CT in 21 patients with
type 1 DM with PDR, 10 patients with NPDR, in 11 patients with normal
fundi, and in 12 normal volunteers without family history of essential
hypertension used as a control group.
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To determine the relationship between PDR and other variables (serum
creatinine, AER, GFR, cholesterol, BP, and
Na+/Li+ CT activity) in the
univariate analyses, binary logistic regression models were used while
controlling for multiple potential cofounders. Subsequently, a multiple
logistic regression analysis was carried out, with PDR as the dependent
variable. In this model,
Na+/Li+ CT (odds ratio
[OR]: 4.7, confidence interval [CI]: 1.2–17.6; P =
0.02), diastolic BP (OR, 3.4; CI, 1.3–9.6; P = 0.018)
and glomerular filtration rate (OR, 5.1; CI, 1.6–17.7;
P = 0.007) were the variables that were maintained in
the equation, indicating that they were the main determinants of the
presence of PDR (Table 2)
.
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Discussion
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We have demonstrated, for the first time, that patients with
type 1 DM and proliferative retinopathy have elevated erythrocyte
Na+/Li+ CT activity. The
more severe retinopathy in these patients with increased
Na+/Li+ CT activity cannot
be attributed either to the duration of diabetes or to the level of
metabolic control as indexed by glycohemoglobin values because these
parameters did not differ between the three group of patients. This
observation is important because it may provide further insight into
the pathogenesis and susceptibility to this complication of
DM.
At present, the nature of the association between PDR and
elevated Na+/Li+ CT is
unclear. We have observed that patients with type 1 DM, PDR, and
elevated Na+/Li+ CT also
have a higher frequency of diabetic nephropathy
(micro/macroalbuminuria). Although this finding represents a
confounding factor, this is not surprising because in these patients a
close association between PDR and micro and macroalbuminuria,
abnormalities associated with elevated
Na+/Li+
CT,3
4
5
has been established by large epidemiologic
studies.12
However, we believe that the increased
Na+/Li+ CT observed in our
patients cannot be accounted for solely by the presence of nephropathy.
In agreement with this hypothesis, the mean
Na+/Li+ CT rates remained
higher in patients with PDR even when patients with micro or
macroalbuminuria were eliminated from the analysis, although the number
of patients in the group with PDR was quite small.
In summary, patients with type 1 DM presenting PDR have a higher
mean erythrocyte Na+/Li+ CT
activity than patients without PDR. Further studies are needed with a
larger number of patients to determine the validity of this preliminary
observation.
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Acknowledgements
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The authors thank the Clinical Pathology Laboratory for the
biochemical analyses, the Department of Nuclear Medicine for
measurement of glomerular filtration rates with use of
51Cr-labeled EDTA, and Stephen Hyslop for editing the
manuscript.
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Footnotes
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Supported by a grant from the FAEP/UNICAMP, Brazil. JMLF is the recipient of a scholarship from CAPES, Brazil.
Submitted for publication April 26, 1999; revised August 9 and December 1, 1999; accepted December 30, 1999.
Commercial relationships policy: N.
Corresponding author: José B. Lopes de Faria, Nephrology Unit, FCM, PO Box 6111, State University of Campinas (UNICAMP), Campinas, SP, 13083-970, Brazil. jblfaria{at}zaz.com.br
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References
|
|---|
-
Canessa, M, Adragna, N, Solomon, H, Connolly, TM, Tosteson, BS, Tosteson, DC (1980) Increased sodium-lithium countertransport in red cell of patients with essential hypertension N Engl J Med 302,772-776[Abstract]
-
Mahnensmith, RL, Aronson, PS (1985) The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathophysiological processes Circ Res 56,773-788[Abstract/Free Full Text]
-
Mangili, R, Bending, JJ, Scott, G, Li, LK, Gupta, A, Viberti, GC (1988) Increased sodium-lithium countertransport activity in red cells of patients with insulin-dependent diabetes and nephropathy N Engl J Med 318,146-150[Abstract]
-
Krolewski, AS, Canessa, M, Warram, JH, et al (1988) Predisposition to hypertension and susceptibility to renal disease in insulin dependent diabetes mellitus N Engl J Med 318,140-145[Abstract]
-
Lopes de Faria, JB, Friedman, R, Tariq, T, Viberti, GC (1992) Prevalence of raised sodium-lithium countertransport activity in type I diabetic patients Kidney Int 41,877-882[Medline][Order article via Infotrieve]
-
Monciotti, CG, Semplicini, A, Morocutti, A, et al (1997) Elevated sodium-lithium countertransport activity in erythrocytes is predictive of the development of microalbuminuria in IDDM Diabetologia 40,654-661[Medline][Order article via Infotrieve]
-
Canessa, M, Zerbini, G, Laffel, LMB (1992) Sodium activation of red cell Na+/Li+ countertransport in diabetes: methodology and controversy J Am Soc Nephrol 3,S41-S49[Abstract]
-
Moss, SE, Klein, R, Klein, BEK (1994) Ocular factors in the incidence and progression of diabetic retinopathy Ophthalmology 101,77-83[Medline][Order article via Infotrieve]
-
Klein, R, Klein, BEK, Moss, SE, Davis, MD, DeMets, DL. (1984) The Wisconsin epidemiological study of diabetic retinopathy. II. Prevalence and risk of diabetic retinopathy when age at diagnosis is less than 30 years Arch Ophthalmol 102,520-526[Abstract/Free Full Text]
-
Kats, J, Zeger, S, Liang, KY (1994) Appropriate statistical methods to account for similarities in binary outcomes between fellow eyes Invest Ophthalmol Vis Sci 35,2461-2465[Abstract/Free Full Text]
-
Lima, PRM, Gontijo, JAR, Lopes de Faria, JB, Costa, FF, Saad, STO (1997) Band 3 Campinas: a novel splicing mutation in the band 3 gene (AE1) associated with hereditary spherocytosis, hyperactivity of Na+/Li+ countertransport and an abnormal renal bicarbonate handling Blood 90,1-9[Free Full Text]
-
Cruickshanks, KJ, Ritter, LL, Klein, R, Moss, SE (1993) The association of microalbuminuria with diabetic retinopathy Ophthalmology 100,862-867[Medline][Order article via Infotrieve]
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Abstract
Introduction
