- Email: [email protected]
Plasma vascular endothelial growth factor in Japanese Type 2 diabetic patients with and without nephropathy
Journal of Diabetes and Its Complications 16 (2002) 386 – 390
Plasma vascular endothelial growth factor in Japanese Type 2 diabetic patients with and without nephropathy K. Shimada, T. Baba*, S. Neugebauer, A. Onozaki, D. Yamada, S. Midorikawa, W. Sato, T. Watanabe Third Department of Internal Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan Received 24 August 2001; received in revised form 20 December 2001; accepted 28 December 2001
Abstract Aim: To determine whether plasma vascular endothelial growth factor (VEGF) level is elevated in Type 2 diabetic patients with an early stage of diabetic nephropathy. Methods: We studied 71 Japanese Type 2 diabetic patients with normal serum creatinine level ( < 100 mmol/l) (age 63.0 [60.3 – 65.6] years old, diabetes duration 15.6 [14.0 – 17.3] years, HbA1c 7.36% [7.06 – 7.66%], mean [95% confidence interval, CI]): normoalbuminuric patients (n = 36); microalbuminuric patients (n = 21); and proteinuric patients (n = 14). Plasma VEGF concentration was measured by a quantitative sandwich enzyme immunoassay technique. Results: Plasma VEGF concentration was not related to the degree of albuminuria: normoalbuminuric patients (25 [13 – 95] ng/l, median [25th – 75th percentile]); microalbuminuric patients (33 [15 – 120] ng/l); and proteinuric patients (54 [17 – 107] ng/l). Plasma VEGF level in patients with retinopathy (25 [15 – 95] ng/l, n = 30) was not elevated as compared to those without retinopathy (53 [14 – 126] ng/l, n = 34). Plasma VEGF tended to correlated negatively with diabetes duration (R’s = .217, P = .0690) and HbA1c (R’s = .221, P = .0647), whereas there was no correlation between plasma VEGF level and age, serum creatinine or urinary albumin to creatinine ratio (ACR) of the patients, respectively. Plasma VEGF level in the group of patients with HbA1c equal to or below the median ( < 7.2%) was significantly higher than that in the group of patients with HbA1c above the median ( > 7.2%) ( P < .05). Conclusions: The results suggested that Type 2 diabetic patients with microalbuminuria and those with retinopathy are not necessarily associated with an elevation of circulating plasma VEGF concentration. Plausible association between plasma VEGF level and glycemic control remains to be seen. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Japanese; Nephropathy; Retinopathy; Type 2 diabetes; Vascular endothelial growth factor (VEGF)
1. Introduction Diabetic nephropathy is a syndrome that is characterized by persistent proteinuria, hypertension, relentless decline in glomerular function, and increased cardiovascular morbidity (Borch-Johnsen & Kreiner, 1987). Diabetic patients in Japan, mostly due to Type 2 diabetes, are now estimated to be over 7 million according to the governmental survey reported in 1999 (Ministry of Health, Labor, and Welfare of Japan, 2000), and diabetic nephropathy has become the most common cause of end-stage renal failure in this country (Statistics Committee of the Japanese Society for Dialysis Therapy, 1993). Hyperglycemia, intraglomerular hypertension, and genetic predisposition are assumed to * Corresponding author. Tel.: +81-24-548-2111x2322, 2325; fax: +8124-548-3044. E-mail address: [email protected] (T. Baba).
play key roles in the development and progression of diabetic nephropathy (Cooper, 1998). Nevertheless, the precise mechanism(s) behind the development of nephropathy is yet to be determined. Recent evidences suggest that vascular endothelial growth factor (VEGF), a cytokine that acts as an endothelial cell mitogen and induces microvascular permeability (Leung, Cachianes, Kuang, Goeddel, & Ferrara, 1989), is causally involved in the development of diabetic retinopathy (Aiello et al., 1994; Clermont, Aiello, Mori, Aiello, & Bursell, 1997). Both VEGF and the two forms of VEGF receptors (VEGFR-1 and VEGFR-2) are expressed in kidney (Cooper et al., 1999; Simon et al., 1995). Increased renal VEGF mRNA and glomerular VEGF immunoreactivity were reported with Otsuka Long – Evans Tokushima Fatty (OLETF) rats (Tsuchida, Makita, Yamagishi, et al., 1999). Neutralizing antibody of VEGF was shown to abolish diabetes-associated hyperfiltration and attenuated
1056-8727/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 5 6 - 8 7 2 7 ( 0 2 ) 0 0 1 6 2 - 9
K. Shimada et al. / Journal of Diabetes and Its Complications 16 (2002) 386–390
387
Table 1 Clinical characteristics of patients
Number of patients Age (years) Known diabetes duration (years) HbA1c (%) Hypertension (no/yes)
Normoalbuminuria
Microalbuminuria
Proteinuria
36 61.0 [56.7 – 65.3] 14.5 [12.5 – 16.7] 7.1 [6.6 – 7.5] 15/16
21 65.9 [61.2 – 70.5] 17.1 [13.1 – 20.5] 7.6 [7.1 – 8.1] 5/14
14 63.5 [58.5 – 68.4] 15.9 [11.4 – 20.4] 7.7 [6.9 – 8.6] 4/9
Data are shown as means and [95% confidence intervals].
the increase in urinary albumin excretion in streptozotocininduced diabetic rats (De Vriese et al., 2001). Hypoxia, high glucose concentration, angiotensin II, and various growth factors and cytokines have been shown to stimulate VEGF production (Flyvbjerg, 2000). Chiarelli et al. (2000) recently observed that plasma VEGF is elevated in prepubertal and pubertal children with Type 1 diabetes mellitus. Similarly, Hovind, Tarnow, Oestergaard, and Parving (2000) observed an elevated VEGF in male Type 1 diabetic patients with nephropathy. These findings suggest that VEGF may be involved in the pathogenesis of diabetic nephropathy. With this background, we measured the plasma levels of VEGF in Japanese Type 2 diabetic patients to elucidate whether an elevation of plasma VEGF level would be observed in the early stage of nephropathy.
athy) and those with liver disease and heart failure are not included in the study. Proteinuric patients without diabetic retinopathy were excluded from the study. The patients were recruited to constitute three groups: normoalbuminuric patients (urinary albumin to creatinine ratio [ACR], < 30 mg/g creatinine, n = 36); microalbuminuric patients (ACR 30– 300 mg/g creatinine, n = 21); and proteinuric patients (ACR > 300 mg/g creatinine, n = 14). The study was performed in accordance with the Second Helsinki Declaration and was approved by the ethical committee of Fukushima Medical University. The informed consent was given to each patient before the study. 2.2. Methods
We studied 71 Japanese Type 2 diabetic patients (40 women and 31 men, age 63.0 [60.3 – 65.6], mean [95% confidence interval, CI]) after an informed consent was given. The patients were chosen from those regularly attending the diabetes clinic of the Fukushima Medical University Hospital on the bases of age ( > 40 years old), known duration of diabetes ( > 5 years), normal serum creatinine level ( < 100 mmol/l), and urinary albumin excretion as described later. Patients with any clinical or laboratory evidence of other kidney disease (besides diabetic nephrop-
Venous blood was sampled in the morning from an antecubital vein into EDTA tubes with minimal stasis, with patients in a sitting position. Centrifugation was done at 1500 g for 15 min within an hour after the samplings, and plasma was stored at 20 C until the assay, which was done within 4 weeks after sampling. Plasma VEGF concentration was determined by a quantitative sandwich enzyme immunoassay technique (Quantikine, R&D Systems, Minneapolis, MN, USA). The immunoassay kit was designed to measure VEGF165 among the several isoforms of VEGF. Urinary albumin concentration was measured using a commercially available kit (Shionoria Albumin, Shionogi & Co., Osaka, Japan). Serum and urinary creatinine levels were measured by enzymatic method adapted to autoanalyzers (Hitachi 7170 and 7600, Hitachi, Japan). HbA1c was determined by high-performance liquid chromatography
Fig. 1. Plasma VEGF levels (ng/ml) in Japanese Type 2 diabetic patients with normoalbuminuria (n = 36, A), microalbuminuria (n = 21, B), and nephropathy (n = 14, C).
Fig. 2. Plasma VEGF levels (ng/ml) in Japanese Type 2 diabetic patients with (n = 30, yes) and without retinopathy (n = 34, no).
2. Patients and methods 2.1. Patients
388
K. Shimada et al. / Journal of Diabetes and Its Complications 16 (2002) 386–390
(Hi-AUTO HA-8150, ARKRAY, Kyoto, Japan) (normal range 3.8– 5.8%). Diabetic retinopathy and its stage were diagnosed by an ophthalmologist of the Fukushima Medical University. Patients with background, preproliferative, and proliferative retinal changes are diagnosed to have retinopathy. Blood pressure was measured with a standard sphygmomanometer at the right arm of patients in a sitting position after 5 min of rest. Hypertension was diagnosed when the mean systolic blood pressure of three consecutive visits was 140 mm Hg and/or the mean diastolic blood pressure was 90 mm Hg, or when the patient was under any pharmacological antihypertensive treatment. Ischemic heart disease was diagnosed electrocardiographically. 2.3. Statistical analyses The differences among the groups were tested by the Kruskal –Wallis rank test or Wilcoxon’s rank sum test, where appropriate. Correlations between two variables were calculated as Spearman’s rank correlation coefficient. A P value < .05 was considered to be significant. The data for age, known diabetes duration, and HbA1c are shown as means (and 95% CI) (Table 1). Because of the skewed distribution of plasma VEGF levels, plasma VEGF concentration in each group was presented as median (and 25th – 75th percentiles).
3. Results There was no statistical difference in plasma VEGF concentrations among the three groups ( P = .7690): 25 [13 – 95] ng/l in the normoalbuminurics; 33 [16 – 120] ng/l in the microalbuminurics; and 54 [17 – 107] ng/l in the proteinurics, median [25th – 75th percentiles] (Fig. 1). Similarly, no elevation in plasma VEGF was observed in patients with retinopathy (25 [15 – 95] ng/l) as compared with those without retinopathy (53 [14 – 126] ng/l, P = .5141) (Fig. 2). There was a trend toward the observation that plasma VEGF concentration correlated negatively with diabetes duration (R’s = .217, P = .0690) and HbA1c (R’s = .221, P = .0647). Plasma VEGF level in the group of patients with HbA1c equal to or below the median ( < 7.2%) was significantly higher (57 [15 – 169] ng/l) than that in the group of patients with HbA1c above the median ( >7.2%) (20 [12 – 61] ng/dl, P < .05). Plasma VEGF conTable 2 Plasma VEGF levels in subgroups
Gender
Smoking Ischemic heart disease
Women
Men
P value
47 (12.5 – 110)
25 (12.5 – 75)
.3798
Yes
No
P value
20.5 (12.5 – 70) 29 (12.5 – 108)
33 (14 – 95) 51 (14.3 – 95)
.2765 .5536
Data are shown as medians and (25 – 75 percentiles).
centration was not correlated with age, body mass index, serum creatinine level, ACR, systolic and diastolic blood pressures, total serum cholesterol, and high-density lipoprotein cholesterol levels of the patients. Gender and history of ischemic heart disease was not associated with plasma VEGF level (Table 2).
4. Discussion Plasma VEGF level did not differ among the three groups of patients with different stages of nephropathy, although we cannot totally exclude the possibility of type II statistic error due to rather a small number of study patients. Similarly, Wasada, Kawahara, Katsumori, Naruse, and Omori (1998) observed no difference in plasma immunoreactive VEGF level in Japanese Type 2 diabetic patients with and without nephropathy. They observed, however, that the prevalence of measurable plasma VEGF was insignificantly increased in parallel with a degree of albuminuria (Wasada et al., 1998). These observations in Japanese Type 2 diabetic patients are in agreement with a previous observation by Hovind et al. (2000) in female Type 1 diabetic patients, but may differ from that in male patients (Hovind et al., 2000) and in adolescents and young adults with Type 1 diabetes with microvascular complications (Chiarelli et al., 2000). The reason(s) of divergent observation is unclear. One possible reason might be the difference in patients’ selection, as we excluded the patients with elevated serum creatinine level. Hovind et al. (2000) defined diabetic nephropathy as having persistent albuminuria of > 300 mg/day. Nevertheless, their table for patients’ characteristics shows that their nephropathic patients (defined as those with urinary albumin excretion rate [AER] >300 mg/24 h) actually had a wider range of AER (ranging from 16 to 14; 545 mg/24 h) (Hovind et al., 2000). This means that some normoalbuminuric (AER 30 mg/24 h) and microalbuminuric (AER 30 – 300 mg/24 h) patients were also included in the nephropathic group, together with those with end-stage renal failure (serum creatinine ranged from 54 to 684 mmol/l) (Hovind et al., 2000). Therefore, the results are not adequately reflecting the plasma VEGF level of different stages of nephropathy. A recent study with a larger number of Type 1 diabetic patients could not demonstrate the difference in plasma VEGF level in patients with and without microalbuminuria (Chaturvedi et al., 2001). These results, together with ours, may suggest that circulating plasma VEGF level is not strongly correlated with renal microvascular disease in diabetic patients. We observed similar plasma level of VEGF in patients with and without established diabetic retinopathy. Chaturvedi et al. (2001) found no association between plasma VEGF level and retinopathy status in a larger number of Type 1 diabetic patients. Similarly, our result is in accordance with the previous observations in Type 1 (Hovind et al., 2000) and Type 2 diabetic patients (Wasada et al.,
K. Shimada et al. / Journal of Diabetes and Its Complications 16 (2002) 386–390
1998), in whom the investigators found no difference in plasma VEGF levels and the severity of retinopathy or progression of retinopathy. On the other hand, local VEGF is shown to be elevated and assumed to play a pivotal role in aggravating diabetic retinopathy. It is likely that the increase in local VEGF production in ocular fluid (Aiello et al., 1994) may not be reflected as an increase in circulating plasma VEGF level. This speculation is strengthened by the observation by Burgos et al. (1997), who found no correlation between plasma and vitreous VEGF concentrations in same patients. Chiarelli et al. (2000) reported that an elevated plasma VEGF level in diabetic children and adolescents with poor glycemic control (HbA1c>10%) decreased after 2 years of tight metabolic control (HbA1c < 7.0%), suggesting the possible influence of long-term glycemic control on plasma VEGF level. In contrast, we observed a trend of an inverse relationship between glycemic status and plasma VEGF level. The median HbA1c level was 7.2% in the present study, which is comparatively a better metabolic control as compared with the studies by Chiarelli et al. (2000) and Hovind et al. (2000) (i.e., 8.5 –9.6%), whereas the reference normal interval of HbA1c in our institute is substantially lower than these clinics. Hypoglycemia is not likely the cause of different observation, because the group of patients with HbA1c level below the median in our study was not necessarily associated with an increased episode of hypoglycemia, which is a known stimulant of VEGF production (Brooks, Gu, Kaufmann, Marcus, & Caldwell, 1998; Park et al., 2001). An association between age and plasma VEGF level reported in Type 1 diabetic children (Chiarelli et al., 2000) was not observed in our study with adult Type 2 diabetic patients. It is likely that pubertal development is related to the increase in VEGF level, as androgens (Sordello, Bertarand, & Planet, 1998), estradiol (Suzuma et al., 1999), and IGF-1 (Punglia et al., 1997), which are elevated in pubertal period and shown to upregulate VEGF. It remains to be seen whether vascular endothelial function would be more responsive to various stimuli in young subjects with short diabetes duration than elderly patients with long diabetes. In conclusion, the results suggest that circulating plasma VEGF level is not associated with microalbuminuria and proteinuria in Japanese Type 2 diabetic patients.
Acknowledgments The authors are thankful to Dr. Shigeatsu Hashimoto, and Ms. Atsuko Hashimoto, Third Department of Internal Medicine, for their cooperation to the study.
References Aiello, L. P., Avery, R. L., Arrigg, P. G., Keyt, B. A., Jampel, H. D., Shah, S. T., Pasquale, L. R., Thieme, H., Iwamoto, M. A., Park, J. E., Nguyen, H. V.,
389
Aiello, L. M., Ferrara, N., & King, G. L. (1994). Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. New England Journal of Medicine, 331, 1480 – 1487. Borch-Johnsen, K., & Kreiner, S. (1987). Proteinuria: value as predictor of cardiovascular mortality in insulin dependent diabetes mellitus. British Medical Journal, 294, 1651 – 1654. Brooks, S. E., Gu, X., Kaufmann, P. M., Marcus, D. M., & Caldwell, R. B. (1998). Modulation of VEGF production by pH and glucose in retinal Muller cells. Current Eye Research, 17, 875 – 882. Burgos, R., Simo, R., Audi, L., Mateo, C., Mesa, J., Garcia-Ramirez, M., & Carrascosa, A. (1997). Vitreous levels of vascular endothelial growth factor are not influenced by its serum concentrations in diabetic retinopathy. Diabetologia, 40, 1107 – 1109. Chaturvedi, N., Fuller, J. H., Pokrast, F., Rottiers, R., Papazoglou, N., Aiello, L. P., & The EUCLID Study Group. (2001). Circulating plasma vascular endothelial growth factor and microvascular complications of Type 1 diabetes mellitus: the influence of ACE inhibition. Diabetic Medicine, 18, 288 – 294. Chiarelli, F., Spagnolit, A., Basciani, F., Tumini, S., Mezzetti, A., Cipollone, F., Cuccurullo, F., Morgese, G., & Verrotti, A. (2000). Vascular endothelial growth factor (VEGF) in children, adolescents and young adults with Type 1 diabetes mellitus: relation to glycaemic control and microvascular complications. Diabetic Medicine, 17, 650 – 656. Clermont, A. C., Aiello, L. P., Mori, F., Ailello, L. M., & Bursell, S. E. (1997). Vascular endothelial growth factor and severity of non-proliferative diabetic retinopathy mediate retinal haemodynamics in vivo: a potential role for vascular endothelial growth factor in the progression of nonproliferative diabetic retinopathy. American Journal of Opthalmology, 124, 433 – 446. Cooper, M. E. (1998). Pathogenesis, prevention, and treatment of diabetic nephropathy. Lancet, 352, 213 – 219. Cooper, M. E., Vranes, D., Youssef, S., Stacker, S. A., Cox, A. J., Rizkalla, B., Casley, D. J., Bach, L. A., Kelly, D. J., & Gilbert, R. E. (1999). Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes. Diabetes, 48, 2229 – 2239. De Vriese, A., Tilton, R., Elger, M., Stephan, C. C., Kriz, W., & Lameire, N. H. (2001). Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. Journal of the American Society of Nephrology, 12, 993 – 1000. Flyvbjerg, A. (2000). Putative pathophysiological role of growth factors and cytokines in experimental diabetic kidney disease. Diabetologia, 43, 1205 – 1223. Hovind, P., Tarnow, L., Oestergaard, P. B., & Parving, H.-H. (2000). Elevated vascular endothelial growth factor in type 1 diabetic patients with diabetic nephropathy. Kidney International, 57, S56 – S61. Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V., & Ferrara, N. (1989). Vascular endothelial growth factor is a secreted angiogenic mitogen. Science, 246, 1306 – 1309. Ministry of Health, Labor, and Welfare of Japan, Part 2, Chapter 6, Section 4. Promoting lifelong health and regional health. 1998 – 1999 Social security and national life. Annual report on health and welfare, vol. 1. Ministry of Health, Labor and Welfare, Tokyo, 2000. Park, S. H., Kim, K. W., Lee, Y. S., Baek, J. H., Kim, M. S., Lee, Y. M., Lee, M. S., & Kim, Y. J. (2001). Hypolgycemia-induced VEGF expression is mediated by intravcellular Ca2+ and protein kinase C signaling pathway in HepG2 human hepatoblastoma cells. International Journal of Molecular Medicine, 7, 91 – 96. Punglia, R. S., Lu, M., Hsu, J., Kuroki, M., Tolentino, M. J., Keough, K., Levy, A. P., Levy, N. S., Goldberg, M. A., D’Amato, R. J., & Adamis, A. P. (1997). Regulation of vascular endothelial growth factor expression by insulin-like growth factor I. Diabetes, 46, 1619 – 1626. Simon, M., Grone, H. J., Johren, O., Kullmer, J., Plate, K. H., Risau, W., & Fuchs, E. (1995). Expression of vascular endothelial growth factor and its receptors in human renal oncogenesis and in adult kidney. American Journal of Physiology, 268, F240 – F250.
390
K. Shimada et al. / Journal of Diabetes and Its Complications 16 (2002) 386–390
Sordello, S., Bertarand, N., & Planet, J. (1998). Vascular endothelial growth factor is up-regulated in vitro and in vivo by androgens. Biochemical and Biophysical Research Communications, 251, 287 – 290. Statistics Committee of the Japanese Society for Dialysis Therapy. (1993). The chronic dialysis patients registry in Japan — 1991. Journal of the Japanese Society for Dialysis Therapy, 26, 17 – 23. Suzuma, I., Mandai, M., Takagi, H., Suzuma, K., Otani, A., Oh, H., Kobayashi, K., & Honda, Y. (1999). 17 Beta-estradiol increases VEGF receptor-2 and promotes DNA synthesis in retinal microvascular
endothelial cells. Investigative Ophthalmology & Visual Science, 40, 2122 – 2129. Tsuchida, K., Makita, Z., Yamagishi, S., Atsumi, T., Miyoshi, H., Obara, S., Ishida, M., Ishikawa, S., Yasumura, K., & Koike, T. (1999). Suppression of transforming growth factor and vascular endothelial growth factor in diabetic nephropathy in rats by a novel advanced glycation end product inhibitor, OPB-9195. Diabetologia, 42, 579 – 588. Wasada, T., Kawahara, R., Katsumori, K., Naruse, M., & Omori, Y. (1998). Plasma concentration of immunoreactive vascular endothelial growth factor and its relation to smoking. Metabolism, 47, 27 – 30.
Plasma vascular endothelial growth factor in Japanese Type 2 diabetic patients with and without nephropathy K. Shimada, T. Baba*, S. Neugebauer, A. Onozaki, D. Yamada, S. Midorikawa, W. Sato, T. Watanabe Third Department of Internal Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan Received 24 August 2001; received in revised form 20 December 2001; accepted 28 December 2001
Abstract Aim: To determine whether plasma vascular endothelial growth factor (VEGF) level is elevated in Type 2 diabetic patients with an early stage of diabetic nephropathy. Methods: We studied 71 Japanese Type 2 diabetic patients with normal serum creatinine level ( < 100 mmol/l) (age 63.0 [60.3 – 65.6] years old, diabetes duration 15.6 [14.0 – 17.3] years, HbA1c 7.36% [7.06 – 7.66%], mean [95% confidence interval, CI]): normoalbuminuric patients (n = 36); microalbuminuric patients (n = 21); and proteinuric patients (n = 14). Plasma VEGF concentration was measured by a quantitative sandwich enzyme immunoassay technique. Results: Plasma VEGF concentration was not related to the degree of albuminuria: normoalbuminuric patients (25 [13 – 95] ng/l, median [25th – 75th percentile]); microalbuminuric patients (33 [15 – 120] ng/l); and proteinuric patients (54 [17 – 107] ng/l). Plasma VEGF level in patients with retinopathy (25 [15 – 95] ng/l, n = 30) was not elevated as compared to those without retinopathy (53 [14 – 126] ng/l, n = 34). Plasma VEGF tended to correlated negatively with diabetes duration (R’s = .217, P = .0690) and HbA1c (R’s = .221, P = .0647), whereas there was no correlation between plasma VEGF level and age, serum creatinine or urinary albumin to creatinine ratio (ACR) of the patients, respectively. Plasma VEGF level in the group of patients with HbA1c equal to or below the median ( < 7.2%) was significantly higher than that in the group of patients with HbA1c above the median ( > 7.2%) ( P < .05). Conclusions: The results suggested that Type 2 diabetic patients with microalbuminuria and those with retinopathy are not necessarily associated with an elevation of circulating plasma VEGF concentration. Plausible association between plasma VEGF level and glycemic control remains to be seen. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Japanese; Nephropathy; Retinopathy; Type 2 diabetes; Vascular endothelial growth factor (VEGF)
1. Introduction Diabetic nephropathy is a syndrome that is characterized by persistent proteinuria, hypertension, relentless decline in glomerular function, and increased cardiovascular morbidity (Borch-Johnsen & Kreiner, 1987). Diabetic patients in Japan, mostly due to Type 2 diabetes, are now estimated to be over 7 million according to the governmental survey reported in 1999 (Ministry of Health, Labor, and Welfare of Japan, 2000), and diabetic nephropathy has become the most common cause of end-stage renal failure in this country (Statistics Committee of the Japanese Society for Dialysis Therapy, 1993). Hyperglycemia, intraglomerular hypertension, and genetic predisposition are assumed to * Corresponding author. Tel.: +81-24-548-2111x2322, 2325; fax: +8124-548-3044. E-mail address: [email protected] (T. Baba).
play key roles in the development and progression of diabetic nephropathy (Cooper, 1998). Nevertheless, the precise mechanism(s) behind the development of nephropathy is yet to be determined. Recent evidences suggest that vascular endothelial growth factor (VEGF), a cytokine that acts as an endothelial cell mitogen and induces microvascular permeability (Leung, Cachianes, Kuang, Goeddel, & Ferrara, 1989), is causally involved in the development of diabetic retinopathy (Aiello et al., 1994; Clermont, Aiello, Mori, Aiello, & Bursell, 1997). Both VEGF and the two forms of VEGF receptors (VEGFR-1 and VEGFR-2) are expressed in kidney (Cooper et al., 1999; Simon et al., 1995). Increased renal VEGF mRNA and glomerular VEGF immunoreactivity were reported with Otsuka Long – Evans Tokushima Fatty (OLETF) rats (Tsuchida, Makita, Yamagishi, et al., 1999). Neutralizing antibody of VEGF was shown to abolish diabetes-associated hyperfiltration and attenuated
1056-8727/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 5 6 - 8 7 2 7 ( 0 2 ) 0 0 1 6 2 - 9
K. Shimada et al. / Journal of Diabetes and Its Complications 16 (2002) 386–390
387
Table 1 Clinical characteristics of patients
Number of patients Age (years) Known diabetes duration (years) HbA1c (%) Hypertension (no/yes)
Normoalbuminuria
Microalbuminuria
Proteinuria
36 61.0 [56.7 – 65.3] 14.5 [12.5 – 16.7] 7.1 [6.6 – 7.5] 15/16
21 65.9 [61.2 – 70.5] 17.1 [13.1 – 20.5] 7.6 [7.1 – 8.1] 5/14
14 63.5 [58.5 – 68.4] 15.9 [11.4 – 20.4] 7.7 [6.9 – 8.6] 4/9
Data are shown as means and [95% confidence intervals].
the increase in urinary albumin excretion in streptozotocininduced diabetic rats (De Vriese et al., 2001). Hypoxia, high glucose concentration, angiotensin II, and various growth factors and cytokines have been shown to stimulate VEGF production (Flyvbjerg, 2000). Chiarelli et al. (2000) recently observed that plasma VEGF is elevated in prepubertal and pubertal children with Type 1 diabetes mellitus. Similarly, Hovind, Tarnow, Oestergaard, and Parving (2000) observed an elevated VEGF in male Type 1 diabetic patients with nephropathy. These findings suggest that VEGF may be involved in the pathogenesis of diabetic nephropathy. With this background, we measured the plasma levels of VEGF in Japanese Type 2 diabetic patients to elucidate whether an elevation of plasma VEGF level would be observed in the early stage of nephropathy.
athy) and those with liver disease and heart failure are not included in the study. Proteinuric patients without diabetic retinopathy were excluded from the study. The patients were recruited to constitute three groups: normoalbuminuric patients (urinary albumin to creatinine ratio [ACR], < 30 mg/g creatinine, n = 36); microalbuminuric patients (ACR 30– 300 mg/g creatinine, n = 21); and proteinuric patients (ACR > 300 mg/g creatinine, n = 14). The study was performed in accordance with the Second Helsinki Declaration and was approved by the ethical committee of Fukushima Medical University. The informed consent was given to each patient before the study. 2.2. Methods
We studied 71 Japanese Type 2 diabetic patients (40 women and 31 men, age 63.0 [60.3 – 65.6], mean [95% confidence interval, CI]) after an informed consent was given. The patients were chosen from those regularly attending the diabetes clinic of the Fukushima Medical University Hospital on the bases of age ( > 40 years old), known duration of diabetes ( > 5 years), normal serum creatinine level ( < 100 mmol/l), and urinary albumin excretion as described later. Patients with any clinical or laboratory evidence of other kidney disease (besides diabetic nephrop-
Venous blood was sampled in the morning from an antecubital vein into EDTA tubes with minimal stasis, with patients in a sitting position. Centrifugation was done at 1500 g for 15 min within an hour after the samplings, and plasma was stored at 20 C until the assay, which was done within 4 weeks after sampling. Plasma VEGF concentration was determined by a quantitative sandwich enzyme immunoassay technique (Quantikine, R&D Systems, Minneapolis, MN, USA). The immunoassay kit was designed to measure VEGF165 among the several isoforms of VEGF. Urinary albumin concentration was measured using a commercially available kit (Shionoria Albumin, Shionogi & Co., Osaka, Japan). Serum and urinary creatinine levels were measured by enzymatic method adapted to autoanalyzers (Hitachi 7170 and 7600, Hitachi, Japan). HbA1c was determined by high-performance liquid chromatography
Fig. 1. Plasma VEGF levels (ng/ml) in Japanese Type 2 diabetic patients with normoalbuminuria (n = 36, A), microalbuminuria (n = 21, B), and nephropathy (n = 14, C).
Fig. 2. Plasma VEGF levels (ng/ml) in Japanese Type 2 diabetic patients with (n = 30, yes) and without retinopathy (n = 34, no).
2. Patients and methods 2.1. Patients
388
K. Shimada et al. / Journal of Diabetes and Its Complications 16 (2002) 386–390
(Hi-AUTO HA-8150, ARKRAY, Kyoto, Japan) (normal range 3.8– 5.8%). Diabetic retinopathy and its stage were diagnosed by an ophthalmologist of the Fukushima Medical University. Patients with background, preproliferative, and proliferative retinal changes are diagnosed to have retinopathy. Blood pressure was measured with a standard sphygmomanometer at the right arm of patients in a sitting position after 5 min of rest. Hypertension was diagnosed when the mean systolic blood pressure of three consecutive visits was 140 mm Hg and/or the mean diastolic blood pressure was 90 mm Hg, or when the patient was under any pharmacological antihypertensive treatment. Ischemic heart disease was diagnosed electrocardiographically. 2.3. Statistical analyses The differences among the groups were tested by the Kruskal –Wallis rank test or Wilcoxon’s rank sum test, where appropriate. Correlations between two variables were calculated as Spearman’s rank correlation coefficient. A P value < .05 was considered to be significant. The data for age, known diabetes duration, and HbA1c are shown as means (and 95% CI) (Table 1). Because of the skewed distribution of plasma VEGF levels, plasma VEGF concentration in each group was presented as median (and 25th – 75th percentiles).
3. Results There was no statistical difference in plasma VEGF concentrations among the three groups ( P = .7690): 25 [13 – 95] ng/l in the normoalbuminurics; 33 [16 – 120] ng/l in the microalbuminurics; and 54 [17 – 107] ng/l in the proteinurics, median [25th – 75th percentiles] (Fig. 1). Similarly, no elevation in plasma VEGF was observed in patients with retinopathy (25 [15 – 95] ng/l) as compared with those without retinopathy (53 [14 – 126] ng/l, P = .5141) (Fig. 2). There was a trend toward the observation that plasma VEGF concentration correlated negatively with diabetes duration (R’s = .217, P = .0690) and HbA1c (R’s = .221, P = .0647). Plasma VEGF level in the group of patients with HbA1c equal to or below the median ( < 7.2%) was significantly higher (57 [15 – 169] ng/l) than that in the group of patients with HbA1c above the median ( >7.2%) (20 [12 – 61] ng/dl, P < .05). Plasma VEGF conTable 2 Plasma VEGF levels in subgroups
Gender
Smoking Ischemic heart disease
Women
Men
P value
47 (12.5 – 110)
25 (12.5 – 75)
.3798
Yes
No
P value
20.5 (12.5 – 70) 29 (12.5 – 108)
33 (14 – 95) 51 (14.3 – 95)
.2765 .5536
Data are shown as medians and (25 – 75 percentiles).
centration was not correlated with age, body mass index, serum creatinine level, ACR, systolic and diastolic blood pressures, total serum cholesterol, and high-density lipoprotein cholesterol levels of the patients. Gender and history of ischemic heart disease was not associated with plasma VEGF level (Table 2).
4. Discussion Plasma VEGF level did not differ among the three groups of patients with different stages of nephropathy, although we cannot totally exclude the possibility of type II statistic error due to rather a small number of study patients. Similarly, Wasada, Kawahara, Katsumori, Naruse, and Omori (1998) observed no difference in plasma immunoreactive VEGF level in Japanese Type 2 diabetic patients with and without nephropathy. They observed, however, that the prevalence of measurable plasma VEGF was insignificantly increased in parallel with a degree of albuminuria (Wasada et al., 1998). These observations in Japanese Type 2 diabetic patients are in agreement with a previous observation by Hovind et al. (2000) in female Type 1 diabetic patients, but may differ from that in male patients (Hovind et al., 2000) and in adolescents and young adults with Type 1 diabetes with microvascular complications (Chiarelli et al., 2000). The reason(s) of divergent observation is unclear. One possible reason might be the difference in patients’ selection, as we excluded the patients with elevated serum creatinine level. Hovind et al. (2000) defined diabetic nephropathy as having persistent albuminuria of > 300 mg/day. Nevertheless, their table for patients’ characteristics shows that their nephropathic patients (defined as those with urinary albumin excretion rate [AER] >300 mg/24 h) actually had a wider range of AER (ranging from 16 to 14; 545 mg/24 h) (Hovind et al., 2000). This means that some normoalbuminuric (AER 30 mg/24 h) and microalbuminuric (AER 30 – 300 mg/24 h) patients were also included in the nephropathic group, together with those with end-stage renal failure (serum creatinine ranged from 54 to 684 mmol/l) (Hovind et al., 2000). Therefore, the results are not adequately reflecting the plasma VEGF level of different stages of nephropathy. A recent study with a larger number of Type 1 diabetic patients could not demonstrate the difference in plasma VEGF level in patients with and without microalbuminuria (Chaturvedi et al., 2001). These results, together with ours, may suggest that circulating plasma VEGF level is not strongly correlated with renal microvascular disease in diabetic patients. We observed similar plasma level of VEGF in patients with and without established diabetic retinopathy. Chaturvedi et al. (2001) found no association between plasma VEGF level and retinopathy status in a larger number of Type 1 diabetic patients. Similarly, our result is in accordance with the previous observations in Type 1 (Hovind et al., 2000) and Type 2 diabetic patients (Wasada et al.,
K. Shimada et al. / Journal of Diabetes and Its Complications 16 (2002) 386–390
1998), in whom the investigators found no difference in plasma VEGF levels and the severity of retinopathy or progression of retinopathy. On the other hand, local VEGF is shown to be elevated and assumed to play a pivotal role in aggravating diabetic retinopathy. It is likely that the increase in local VEGF production in ocular fluid (Aiello et al., 1994) may not be reflected as an increase in circulating plasma VEGF level. This speculation is strengthened by the observation by Burgos et al. (1997), who found no correlation between plasma and vitreous VEGF concentrations in same patients. Chiarelli et al. (2000) reported that an elevated plasma VEGF level in diabetic children and adolescents with poor glycemic control (HbA1c>10%) decreased after 2 years of tight metabolic control (HbA1c < 7.0%), suggesting the possible influence of long-term glycemic control on plasma VEGF level. In contrast, we observed a trend of an inverse relationship between glycemic status and plasma VEGF level. The median HbA1c level was 7.2% in the present study, which is comparatively a better metabolic control as compared with the studies by Chiarelli et al. (2000) and Hovind et al. (2000) (i.e., 8.5 –9.6%), whereas the reference normal interval of HbA1c in our institute is substantially lower than these clinics. Hypoglycemia is not likely the cause of different observation, because the group of patients with HbA1c level below the median in our study was not necessarily associated with an increased episode of hypoglycemia, which is a known stimulant of VEGF production (Brooks, Gu, Kaufmann, Marcus, & Caldwell, 1998; Park et al., 2001). An association between age and plasma VEGF level reported in Type 1 diabetic children (Chiarelli et al., 2000) was not observed in our study with adult Type 2 diabetic patients. It is likely that pubertal development is related to the increase in VEGF level, as androgens (Sordello, Bertarand, & Planet, 1998), estradiol (Suzuma et al., 1999), and IGF-1 (Punglia et al., 1997), which are elevated in pubertal period and shown to upregulate VEGF. It remains to be seen whether vascular endothelial function would be more responsive to various stimuli in young subjects with short diabetes duration than elderly patients with long diabetes. In conclusion, the results suggest that circulating plasma VEGF level is not associated with microalbuminuria and proteinuria in Japanese Type 2 diabetic patients.
Acknowledgments The authors are thankful to Dr. Shigeatsu Hashimoto, and Ms. Atsuko Hashimoto, Third Department of Internal Medicine, for their cooperation to the study.
References Aiello, L. P., Avery, R. L., Arrigg, P. G., Keyt, B. A., Jampel, H. D., Shah, S. T., Pasquale, L. R., Thieme, H., Iwamoto, M. A., Park, J. E., Nguyen, H. V.,
389
Aiello, L. M., Ferrara, N., & King, G. L. (1994). Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. New England Journal of Medicine, 331, 1480 – 1487. Borch-Johnsen, K., & Kreiner, S. (1987). Proteinuria: value as predictor of cardiovascular mortality in insulin dependent diabetes mellitus. British Medical Journal, 294, 1651 – 1654. Brooks, S. E., Gu, X., Kaufmann, P. M., Marcus, D. M., & Caldwell, R. B. (1998). Modulation of VEGF production by pH and glucose in retinal Muller cells. Current Eye Research, 17, 875 – 882. Burgos, R., Simo, R., Audi, L., Mateo, C., Mesa, J., Garcia-Ramirez, M., & Carrascosa, A. (1997). Vitreous levels of vascular endothelial growth factor are not influenced by its serum concentrations in diabetic retinopathy. Diabetologia, 40, 1107 – 1109. Chaturvedi, N., Fuller, J. H., Pokrast, F., Rottiers, R., Papazoglou, N., Aiello, L. P., & The EUCLID Study Group. (2001). Circulating plasma vascular endothelial growth factor and microvascular complications of Type 1 diabetes mellitus: the influence of ACE inhibition. Diabetic Medicine, 18, 288 – 294. Chiarelli, F., Spagnolit, A., Basciani, F., Tumini, S., Mezzetti, A., Cipollone, F., Cuccurullo, F., Morgese, G., & Verrotti, A. (2000). Vascular endothelial growth factor (VEGF) in children, adolescents and young adults with Type 1 diabetes mellitus: relation to glycaemic control and microvascular complications. Diabetic Medicine, 17, 650 – 656. Clermont, A. C., Aiello, L. P., Mori, F., Ailello, L. M., & Bursell, S. E. (1997). Vascular endothelial growth factor and severity of non-proliferative diabetic retinopathy mediate retinal haemodynamics in vivo: a potential role for vascular endothelial growth factor in the progression of nonproliferative diabetic retinopathy. American Journal of Opthalmology, 124, 433 – 446. Cooper, M. E. (1998). Pathogenesis, prevention, and treatment of diabetic nephropathy. Lancet, 352, 213 – 219. Cooper, M. E., Vranes, D., Youssef, S., Stacker, S. A., Cox, A. J., Rizkalla, B., Casley, D. J., Bach, L. A., Kelly, D. J., & Gilbert, R. E. (1999). Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes. Diabetes, 48, 2229 – 2239. De Vriese, A., Tilton, R., Elger, M., Stephan, C. C., Kriz, W., & Lameire, N. H. (2001). Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. Journal of the American Society of Nephrology, 12, 993 – 1000. Flyvbjerg, A. (2000). Putative pathophysiological role of growth factors and cytokines in experimental diabetic kidney disease. Diabetologia, 43, 1205 – 1223. Hovind, P., Tarnow, L., Oestergaard, P. B., & Parving, H.-H. (2000). Elevated vascular endothelial growth factor in type 1 diabetic patients with diabetic nephropathy. Kidney International, 57, S56 – S61. Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V., & Ferrara, N. (1989). Vascular endothelial growth factor is a secreted angiogenic mitogen. Science, 246, 1306 – 1309. Ministry of Health, Labor, and Welfare of Japan, Part 2, Chapter 6, Section 4. Promoting lifelong health and regional health. 1998 – 1999 Social security and national life. Annual report on health and welfare, vol. 1. Ministry of Health, Labor and Welfare, Tokyo, 2000. Park, S. H., Kim, K. W., Lee, Y. S., Baek, J. H., Kim, M. S., Lee, Y. M., Lee, M. S., & Kim, Y. J. (2001). Hypolgycemia-induced VEGF expression is mediated by intravcellular Ca2+ and protein kinase C signaling pathway in HepG2 human hepatoblastoma cells. International Journal of Molecular Medicine, 7, 91 – 96. Punglia, R. S., Lu, M., Hsu, J., Kuroki, M., Tolentino, M. J., Keough, K., Levy, A. P., Levy, N. S., Goldberg, M. A., D’Amato, R. J., & Adamis, A. P. (1997). Regulation of vascular endothelial growth factor expression by insulin-like growth factor I. Diabetes, 46, 1619 – 1626. Simon, M., Grone, H. J., Johren, O., Kullmer, J., Plate, K. H., Risau, W., & Fuchs, E. (1995). Expression of vascular endothelial growth factor and its receptors in human renal oncogenesis and in adult kidney. American Journal of Physiology, 268, F240 – F250.
390
K. Shimada et al. / Journal of Diabetes and Its Complications 16 (2002) 386–390
Sordello, S., Bertarand, N., & Planet, J. (1998). Vascular endothelial growth factor is up-regulated in vitro and in vivo by androgens. Biochemical and Biophysical Research Communications, 251, 287 – 290. Statistics Committee of the Japanese Society for Dialysis Therapy. (1993). The chronic dialysis patients registry in Japan — 1991. Journal of the Japanese Society for Dialysis Therapy, 26, 17 – 23. Suzuma, I., Mandai, M., Takagi, H., Suzuma, K., Otani, A., Oh, H., Kobayashi, K., & Honda, Y. (1999). 17 Beta-estradiol increases VEGF receptor-2 and promotes DNA synthesis in retinal microvascular
endothelial cells. Investigative Ophthalmology & Visual Science, 40, 2122 – 2129. Tsuchida, K., Makita, Z., Yamagishi, S., Atsumi, T., Miyoshi, H., Obara, S., Ishida, M., Ishikawa, S., Yasumura, K., & Koike, T. (1999). Suppression of transforming growth factor and vascular endothelial growth factor in diabetic nephropathy in rats by a novel advanced glycation end product inhibitor, OPB-9195. Diabetologia, 42, 579 – 588. Wasada, T., Kawahara, R., Katsumori, K., Naruse, M., & Omori, Y. (1998). Plasma concentration of immunoreactive vascular endothelial growth factor and its relation to smoking. Metabolism, 47, 27 – 30.