Introduction. ties [13 16]. In addition, [Ca 2+ ]i has been reported to be elevated in diabetic patients [17, 18]. Accordingly,

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1 Nisoldipine improves the impaired erythrocyte deformability correlating with elevated intracellular free calcium-ion concentration and poor glycaemic control in NIDDM J. Fujita, 1 K. Tsuda, 2 T. Takeda, 1 L. Yu, 1 S. Fujimoto, 1 M. Kajikawa, 1 M. Nishimura, 1 N. Mizuno, 1 Y. Hamamoto, 1 E. Mukai, 1 T. Adachi 2 & Y. Seino 1 1 Department of Metabolism and Clinical Nutrition, Kyoto University Graduate School of Medicine and 2 Department of Integrated Human Science, Kyoto University, Kyoto, Japan Aims To explore the mechanisms underlying the impaired erythrocyte deformability (RBC-df ) in diabetic patients, the relationship between erythrocyte intracellular free calcium-ion concentration ([Ca 2+ ]i) and RBC-df, and the effects of Ca 2+ - channel blocker on [Ca 2+ ]i and RBC-df were evaluated. Methods Forty-eight patients with NIDDM and 24 control subjects were enrolled in this study. [Ca 2+ ]i was determined using fura-2, and RBC-df by filtration method expressed as Deformability Index (DI). Erythrocytes were treated with nisoldipine to evaluate the effects of a Ca 2+ -channel blocker. Results [Ca 2+ ]i was significantly higher (82.6 ( ) vs 76.6 ( ) nmol lrbc 1, P<0.001), and DI was significantly lower (0.14 ( ) vs 0.22 ( ), ) in NIDDM than in controls. There was a significant correlation between HbA 1c and [Ca 2+ ]i (r=0.38, ), between HbA 1c and DI (r= 0.51, ), and between [Ca 2+ ]i and DI (r= 0.42, ). Stepwise multiple regression analysis revealed HbA 1c and [Ca 2+ ]i as independent determinants for the impaired RBC-df. Nisoldipine treatment in vitro significantly decreased [Ca 2+ ]i, and significantly improved RBC-df. Conclusions These data indicate that the impaired RBC-df in NIDDM may at least partly be attributed to the elevated [Ca 2+ ]i and poor glycaemic control. In addition, favorable effects of a Ca 2+ -channel blocker on both [Ca 2+ ]i and RBC-df have been demonstrated. Keywords: NIDDM, erythrocyte deformability, intracellular free calcium-ion, Ca 2+ -channel blocker, nisoldipine Introduction ties [13 16]. In addition, [Ca 2+ ]i has been reported to be elevated in diabetic patients [17, 18]. Accordingly, Erythrocyte deformability (RBC-df ) has been proposed one additional engaging working hypothesis revolves to play an important role in the pathogenesis of diabetic around the dysfunction of erythrocyte calcium homoeostasis vascular complications [1, 2]. Many studies have shown in diabetes. that RBC-df is decreased in patients with diabetes In this context, it is important to note that Ca 2+ - mellitus [3 12]. channel blockers suppress Ca 2+ influx into erythrocytes While various mechanisms have been attributed to the on the one hand [19, 20], and improve RBC-df on the reduced RBC-df observed in diabetes, the nature of the other hand [21 24]. Thus, patients with disorders primary defects remains elusive. A number of reports associated with reduced RBC-df, including diabetic have revealed a critical role for maintaining low erythrocyte patients, might benefit clinically not only from the intracellular free calcium-ion concentration ([Ca 2+ ]i) antihypertensive effects of Ca 2+ channel blockers, but levels to maintain normal erythrocyte mechanical proper- also from favourable effects on RBC-df. Therefore, to explore the mechanism underlying the Correspondence: Dr Jun Fujita, Department of Metabolism and Clinical impaired RBC-df in diabetic patients, we have evaluated Nutrition, Kyoto University Graduate School of Medicine, 54 Shogointhe relationship between [Ca 2+ Kawahara-cho, Sakyo-ku, Kyoto, , Japan. ]i and RBC-df in patients Received 30 April 1998, accepted 15 January with NIDDM. Furthermore, the effects of nisoldipine, a 1999 Blackwell Science Ltd Br J Clin Pharmacol, 47,

2 J. Fujita et al. 1,4-dihydropyridine Ca 2+ -channel blocker, on [Ca 2+ ]i heparin-treated tubes. Blood glucose, triglycerides, total and RBC-df have also been investigated. cholesterol, HDL-cholesterol, and HbA 1c were measured. HbA 1c was determined by high-pressure liquid chromatography with a normal range of %, and other Methods measurements were performed by routine laboratory Subjects methods by an auto-analyzer. Erythrocyte count, haemoglobin concentration, Forty-eight patients with NIDDM (23 M; 25 F; aged 30 to haematocrit, MCV, MCH, MCHC, WBC count, and 64 years), randomly selected from the patients attending platelet count were determined by Coulter counter. our outpatient clinic at Kyoto University Hospital, were There was no difference in all of these haematological included in this study (DM group). The criteria of inclusion parameters between control group and DM group (data in this study were as follows: 1) NIDDM diagnosed by not shown). An aliquot of the blood sample was processed WHO criteria (1985), 2) serum creatinine level of less than for the measurements of [Ca 2+ ]i and RBC-df. 130 mmol l 1, 3) no clinical or laboratory evidence of other kidney or renal tract diseases. None of the patients had a past history of cardiovascular disease. Fifteen patients [Ca 2+ ]i measurements were taking Ca 2+ -channel blockers, ten patients ACE Measurement of [Ca 2+ ]i was performed using fura-2 inhibitors, seven patients b-adrenoceptor blockers, and four according to the method reported in detail in our patients diuretics. None of the antihypertensive agents was previous report [25], based on the method described by discontinued or changed in its dosage throughout the study. David-Dufilho et al. [26], with slight modifications. Twenty-four healthy, normotensive volunteers (12 M; and Briefly, erythrocytes were suspended at 1% haematocrit 12 F; aged 28 to 60 years) were enrolled as normal controls in buffer A (123 mmol l 1 NaCl, 5 mmol l 1 KCl, (control group). This study was performed in accordance 1mmol l 1 MgCl 2, 1 mmol l 1 CaCl 2, 10 mmol l 1 with the principles of the Helsinki declaration and approval glucose, 25 mmol l 1 HEPES, ph 7.4 at 37 C), and for the study was given by the Ethics Committee of Kyoto then incubated for 60 min at 37 C with or without University. Written informed consent was given by all 0.5 mmol l 1 fura-2-acetoxymethylester. Erythrocytes subjects. were washed three times with buffer A, and then diluted Classification of nephropathy was as follows. Three to 0.1% haematocrit in buffer B (buffer A contains consecutive, sterile overnight urine collections were additional 1 mmol l 1 MnCl 2 to quench the extracellular performed for measurement of urinary albumin excretion fura-2 signals). Fluorescence was measured by fluorescent rate (AER), using radioimmunoassay. The median value spectrophotometer (Shimadzu RF-5000, Kyoto, Japan) of three specimens was used for classifying the patients in quartz cuvettes. The excitation wavelengths were into three categories: normoalbuminuria; AER 340 nm and 380 nm with 3 nm bandwidth and the <20 mg min 1, microalbuminuria; AER emission wavelength was 500 nm with 10 nm bandwidth. mg min 1, macroalbuminuria; AER >200 mg min 1. [Ca 2+ ]i was calculated according to the equation The patients were diagnosed to have diabetic nephropathy described by Grinkiewicz et al. [27]. Fluorescence when they had microalbuminuria or macroalbuminuria. intensities were calculated by subtraction of the fluorescence Fundus examination was performed by the ophthalmologist of fura-2 unloaded erythrocytes (intrinsic fluores- after mydriasis once a year. The findings were cence) measured at 340 and 380 nm from those of fura-2 graded as: 1) no signs of diabetic retinopathy; 2) simple loaded erythrocytes. The parameters used in the equation retinopathy; or 3) proliferative retinopathy. were determined from fura-2-calcium fluorescence calibration All subjects were weighed in indoor clothing without experiments with EGTA-calcium buffers sim- shoes at every visit, and height was also recorded. Blood ilar to the intracellular conditions (10 mmol l 1 NaCl, pressure ( phase I and V) was measured at every visit 120 mmol l 1 KCl, 0.4 mmol l 1 MgCl 2, 10 mmol l 1 three times in a sitting position after at least 15 min rest glucose, 25 mmol l 1 HEPES, 21 g l 1 polyvinylpyrrolidone, by a standard mercury sphygmomanometer, with cuffs and variable Ca 2+ concentrations). adapted to arm circumference. The median value of the three readings was reported as the value of the visit. Hypertension was diagnosed when the median value of Deformability measurements the three consecutive office visits was above 140 mmhg Determination of RBC-df was performed according to (systolic blood pressure) and/or 90 mmhg (diastolic blood the method described by Brown et al. [28] based on the pressure), or the patient was already on antihypertensive guidelines set by the International Committee for medication. Standardization in Haematology, Expert Panel on Blood Venous blood was collected after an overnight fast into Rheology [29] with slight modifications. At first, whole Blackwell Science Ltd Br J Clin Pharmacol, 47,

3 Nisoldipine improves erythrocyte deformability in NIDDM blood was filtered through cotton wool removed from a In contrast, DI was significantly lower in the DM group leukocyte filter (Imugard 500, Terumo, Tokyo, Japan) than in the control group (Figure 1b; 0.14 ( ) vs [30]. After high-speed centrifugation, plasma and upper ( ); ). In the DM group, there was most layer of erythrocytes were aspirated. The remaining no difference of [Ca 2+ ]i and DI among the subgroups erythrocytes were washed three times with isotonic divided on the basis of nephropathy, retinopathy, PBS (NaCl 8.0 g, KCl 0.2 g, Na 2 HPO 4.12H 2 O 2.9 g, hypertension, or taking a Ca 2+ -channel blocker (data KH 2 PO g, and albumin 5 g up to 1 l, ph=7.4 ). not shown). Washed erythrocytes, aspirated from the middle of the packed erythrocyte column, were resuspended in isotonic PBS to a final concentration of 5%. Virtually no Erythrocyte deformability and glycaemic control contamination of WBC was observed by this method. Univariate regression analyses were applied to the data RBC-df was expressed as a deformability index (DI), obtained in the DM group. There was a significant defined as the time required for 5 ml of PBS to pass positive correlation between HbA 1c and [Ca 2+ ]i through a 5 mm pore filter (Nucleopore, Pleasanton, (Figure 2a;, r=0.38, ). In contrast, there USA) under a constant negative pressure of 20 cm were significant inverse correlations between HbA 1c and H 2 Oat37 C, divided by the time required for 5 ml of DI (Figure 2b;, r= 0.51, ), and between erythrocyte suspension. In this definition, higher DI [Ca 2+ ]i and DI (Figure 2c;, r= 0.42, ). means better deformability. The DI was reported as the There is no significant relationship between blood average of three repeated measurements. pressure, RBC-df, and [Ca 2+ ]i. To elucidate the independently contributing factors to Effects of nisoldipine decreased DI in the DM group, stepwise multiple regression analysis was carried out (Table 2). HbA 1c Nisoldipine treatment in vitro was performed by incubating appeared as the first significant determinant for DI, and the fura-2 loaded erythrocytes, or 5% erythrocyte [Ca 2+ ]i appeared as the second. All the other factors suspension in the respective buffer containing 10 7 evaluated ( gender, age, known duration of diabetes, mol l 1 of nisoldipine (from 10 4 mol l 1 stock solution existence of microalbuminuria or macroalbuminuria, dissolved in ethanol) for 10 minutes at 37 C. Erythrocytes existence of simple or proliferative retinopathy, BMI, treated with 0.01% ethanol alone were used as controls. sbp, dbp, triglycerides, total- and HDL-cholesterol) did Treatment with ethanol alone did not influence the [Ca 2+ ]i and RBC-df. Then the respective erythrocyte suspension was used for the determination of [Ca 2+ ]i and DI. not appear as significant determinants. In this model, 41% of the variance in DI could be explained by these two factors ( P<0.0001). Effects of nisoldipine in vitro Statistical analysis After the in vitro nisoldipine treatment, [Ca 2+ ]i was Data were expressed as median and (range). Statistical significantly decreased from 82.6 ( ) nmol analyses were performed using Wilcoxon s rank-sum l RBC 1 to 80.4 ( ) nmol l RBC 1 (Figure 3a; test (non-matched pairs), Wilcoxon s signed-ranks test, ), and DI was significantly improved from (matched pairs), Spearman s correlation coefficient, and 0.15 ( ) to 0.18 ( ) in the DM group stepwise multiple regression analysis. P values less than (Figure 3b;, ). In addition, there was a 0.05 were considered to be statistically significant. significant positive correlation between the degree of [Ca 2+ ]i decrease and the degree of DI increase (, Results r=0.52, P<0.001). In contrast, there was no significant change in either [Ca 2+ ]i or DI in the control group (n= Clinical characteristics of the subjects are shown in 24, [Ca 2+ ]i: from 76.6 ( ) nmol l RBC 1 to Table 1. Plasma triglycerides and sbp were significantly 76.7 ( ) nmol l RBC 1 ; DI: from 0.22 higher, and HDL-cholesterol was significantly lower in ( ) to 0.24 ( )). the DM group than in the control group. [Ca 2+ ]i and erythrocyte deformability [Ca 2+ ]i was significantly higher in the DM group than in the control group (Figure 1a; 82.6 ( ) nmol lrbc 1 vs 76.6 ( ) nmol lrbc 1 ; P<0.001). Discussion This study is the first to evaluate [Ca 2+ ]i and RBC-df simultaneously in diabetic patients, and has revealed significant correlations of impaired RBC-df with elevated [Ca 2+ ]i and poor glycaemic control. In addition, 1999 Blackwell Science Ltd Br J Clin Pharmacol, 47,

4 [Ca 2+ ]i (nmoll 1 RBC) Deformability index (DI) J. Fujita et al. Table 1 Clinical characteristics of the control subjects and NIDDM patients. Control group DM group n (M/F) 24 (12/12) 48 (23/25) Age (years) 52 (28 60) 54 (30 64) Duration of DM (years) ND 8.2 ( ) BMI (kg m 2 ) ( ) ( ) HbA 1c (%) ND 7.6 ( ) Total cholesterol (mm) 5.4 ( ) 5.2 ( ) HDL-cholesterol (mm) 1.4 ( ) 1.2 ( )* Triglycerides (mm) 1.0 ( ) 1.4 ( )* sbp (mmhg) 120 (90 138) 140 (98 167)* dbp (mmhg) 78 (64 88) 80 (68 96) Incidence of hypertension (%) 0 42 Incidence of retinopathy (%) ND 48 Incidence of nephropathy (%) ND 63 AER (mg min 1 ) ND 48 (7 750) Data are median and (range); ND: not determined; *P<0.05 vs control group. a 95.0 P<0.001 b Controls (24) D M (48) 0.00 Controls (24) Figure 1 a) Comparison of [Ca 2+ ]i between control group and DM group and b) comparison of RBC-df (DI) between control group and DM group. The difference between two groups was analyzed using Wilcoxon s rank-sum test. In brackets number of cases. Horizontal bars represent median. favourable effects of nisoldipine, a dihydropyridine Ca 2+ - is thought to be one of the main pathogenic mechanisms channel blocker, on both [Ca 2+ ]i and RBC-df have of diabetic nephropathy [31]. Moreover, it is generally concurrently been demonstrated for the first time. recognized that haemorheological factors, especially the It has been proposed that impaired RBC-df may play mechanical property of erythrocytes, can play a major an important role in the pathogenesis of diabetic role in governing nutritive tissue perfusion at the level of microangiopathy and macroangiopathy [1, 2]. The the microcirculation [32]. Likewise, impaired RBC-df hypothesis proposes that stiffened erythrocytes would has been found in conditions associated with an increased require raised perfusion pressure to overcome their risk for atherosclerosis, including diabetes mellitus [33, resistance to flow. In the renal circulation, intra- 34], peripheral vascular disease [35], cardiovascular diseases glomerular hypertension would ensue as a result, which [24], and also in cerebrovascular disease [36]. Thus, D M (48) Blackwell Science Ltd Br J Clin Pharmacol, 47,

5 Deformability index (DI) Deformability index (DI) [Ca 2+ ]i (nmoll 1 RBC) Nisoldipine improves erythrocyte deformability in NIDDM a r= HbA1c (%) b HbA1c (%) c r= 0.51 r= [Ca 2+ ]i (nmoll 1 RBC) decreased in patients with diabetes mellitus [3 12], and in animal models of diabetes mellitus [28, 37]. Previously suggested mechanisms underlying reduced RBC-df observed in diabetes include hypoinsulinaemia [5], increased sorbitol concentration [6], increased erythrocyte membrane rigidity caused by glycation of the membrane itself [38], oxidation of spectrin [11], formation of AGEs in erythrocyte [28], and alterations in membrane lipid composition [12]. In accordance with these ideas, several studies, including the present study, have revealed a significant correlation between poor glycaemic control and decreased RBC-df [3, 10, 11]. Although the nature of the primary defects that lead to decreased RBC-df in diabetes mellitus remains elusive, one additional engaging working hypothesis revolves around the dysregulation of erythrocyte calcium homoeostasis. The report by Weed et al. [13] was the first to show increased [Ca 2+ ]i to be associated with decreased erythrocyte membrane deformability. Since then, a number of reports have appeared establishing a critical role for maintaining low [Ca 2+ ]i levels in normal erythrocyte mechanical properties [14 16]. It has also been reported that high [Ca 2+ ]i values were associated with reduced RBC-df in hypertensive patients [19]. Until now, only one group has examined the relationship between [Ca 2+ ]i and erythrocyte rheology in diabetic patients [15]. They reported a significant reverse correlation between [Ca 2+ ]i and erythrocyte membrane fluidity determined by membrane protein lateral mobility. Although some differences in the methodology exist, our present results agree with them in this regard. One hypothesis for the pathogenesis of hypertension relates to an alteration in intracellular ion metabolism [39]. Hypertensive diabetic patients have been shown to have increased intracellular sodium, ph and calcium. This alteration of intracellular milieu may lead to increased vasoconstriction and cellular proliferation which can contribute to an increase in blood pressure and vascular complications. However, there was no significant relationship between blood pressure and RBC-df and [Ca 2+ ]i in our study. This lack of relationship might be due to the antihypertensive treatment. [Ca 2+ ]i has been reported to be elevated in diabetic patients [17, 18]. On the other hand, a decrease in erythrocyte Ca 2+ -ATPase activity has been found in patients with IDDM [40] or NIDDM [41, 42]. It has been suggested that this decreased Ca 2+ -ATPase activity would be due to glycation of the protein itself [43]. Since the plasma membrane Ca 2+ -ATPase is the only system for Figure 2 a) Correlation between HbA 1c and [Ca 2+ ]i, b) correlation between HbA 1c and RBC-df (DI), c) correlation between [Ca 2+ ]i and RBC-df (DI). The relationship between two variables was analyzed using Spearman s correlation coefficient. extruding Ca 2+ from human erythrocytes [44], the elevated [Ca 2+ ]i in diabetic patients might at least partly reduced RBC-df appears to be one factor of importance be attributed to the decreased Ca 2+ -ATPase activity. The also in the pathogenesis of diabetic macroangiopathy. significant positive correlation between HbA 1c and Accumulating evidence has shown that RBC-df is [Ca 2+ ]i presented in our study supports thisidea Blackwell Science Ltd Br J Clin Pharmacol, 47,

6 Deformability index (DI) [Ca 2+ ]i (nmoll 1 RBC) J. Fujita et al. Independent Standard partial Significance Adjusted coefficient variable regression coefficient level of determination Table 2 Stepwise multiple regression analysis with RBC-df (DI) as dependent variable. HbA 1c 0.37 < [Ca 2+ ]i 0.37 < Regression model: DI= HbA 1c [Ca 2+ ]i Independent variables: HbA 1c, [Ca 2+ ]i, gender, age, known duration of diabetes, existence of nephropathy, existence of retinopathy, existence of hypertension, BMI, sbp, dbp, triglycerides, total- and HDL-cholesterol. a b Nisoldipine ( ) Nisoldipine ( ) Nisoldipine (+) Nisoldipine (+) Figure 3 Effects of nisoldipine on [Ca 2+ ]i and b) effects of nisoldipine on RBC-df. [Ca 2+ ]i and DI were measured after the treatment with 10 7 mol l 1 of nisoldipine or with the solvent (0.01% ethanol) alone for 10 min at 37 C. The difference between two groups was analyzed using Wilcoxon s signed-ranks test. Horizontal bars represent median. It has been shown that Ca 2+ -channel blockers can suppress Ca 2+ influx into erythrocytes [19, 20]. We also have reported that nisoldipine blocks the increase of [Ca 2+ ]i in diabetic patients other than those enrolled in this study [45]. On the other hand, it has also been reported that Ca 2+ -channel blockers can improve RBC-df [21 24, 46]. Dihydropyridine binding sites, although somewhat different from those of excitable cells, have been found on human erythrocyte membranes [47]. The existence of a voltage-dependent and dihydropyridine-sensitive calcium influx pathway has also been shown in human erythrocytes [48, 49]. Taking these reported results into consideration, the improved RBC-df caused by Ca 2+ -channel blockers can at least partly be attributed to the effect of decreasing [Ca 2+ ]i. Our present study is the first that has examined the effects of Ca 2+ - channel blockers on [Ca 2+ ]i and RBC-df simultaneously, and that has demonstrated the results supporting this hypothesis. It is of interest that Ca 2+ -channel blockers reduce [Ca 2+ ]i only when the influx of Ca 2+ and [Ca 2+ ]i levels are increased [19, 20]. Furthermore, Ca 2+ -channel blockers are unable to influence the deformability of normal erythrocytes [23, 50]. Our results also agree with these reports. Therefore, only the patients suffering from disorders associated with elevated [Ca 2+ ]i and reduced RBC-df, including diabetic patients, might benefit clinically not only from the antihypertensive effects of the drugs but also from improvement in RBC-df [24]. However, the concentration of nisoldipine used in this study was several to ten times as high as clinically attainable levels, that is around 10 8 mol l 1 [23]. As the clinical dosage of a Ca 2+ -channel blocker did not seem to alter the [Ca 2+ ]i and RBC-df levels in this study, the result is somewhat different from those reported previously [21 23]. These discrepancies might be due to the differences of the Ca 2+ -channel blockers used in the experiments, or of the blood levels of these drugs at the Apart from the effects of Ca 2+ -ATPase, close relationships between intracellular calcium and sodium concen- time of blood sampling. Further investigations are trations have been reported [39]. Unfortunately, we did necessary before conclusions can be drawn about the not measure intracellular sodium concentrations in the clinical relevance of the effects of Ca 2+ -channel blockers present study, and further study will be needed in on RBC-df. this regard. Some studies have found significant correlations Blackwell Science Ltd Br J Clin Pharmacol, 47,

7 Nisoldipine improves erythrocyte deformability in NIDDM between the severity of vascular complications and 9 Jay RH, Jones SL, Hill CE, et al. Blood rheology and decreased RBC-df [3, 4, 7], but other studies, including cardiovascular risk factors in type 1 diabetes: relationship our present study, have not found such correlations [8, with microalbuminuria. Diabet Med 1991; 8: Rendell M, Fox M, Knox S, Lastovica J, Kirchain W, 9]. Since all of these studies are cross-sectional ones, Meiselman HJ. Effects of glycemic control on red cell prospective studies would be required to estimate the deformability determined by using the cell transit time clinical significance of decreased RBC-df in the pathogen- analyzer. J Lab Clin Med 1991; 117: esis of diabetic complications. If diabetic vascular com- 11 Schwartz RS, Madsen JW, Rybicki AC, Nagel RL. plications were caused at least partly by abnormal Oxidation of spectrin and deformability defects in diabetic haemorheology, then the possibility exists that these erythrocytes. Diabetes 1991; 40: might be alleviated or prevented by the agents, including 12 Persson SU, Wohlfart G, Larsson H, Gustafson A. Correlations between fatty acid composition of the Ca 2+ -channel blockers, that enhance RBC-df and erythrocyte membrane and blood rheology data. Scand J Clin improve blood rheology. Longitudinal studies are needed Lab Invest 1996; 56: also in this regard. 13 Weed RI, LaCelle PL, Merrill EW. Metabolic dependence of red cell deformability. J Clin Invest 1969; 48: Shiga T, Sekiya M, Maeda N, Kon K, Okazaki M. Cell agedependent changes in deformability and calcium This study was supported in part by Research Grants from the accumulation of human erythrocytes. 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