Hideyuki Yoshizumi

Association between a polymorphism in the angiotensin-converting enzyme gene and microvascular complications in Japanese patients with NIDDM

SummaryThe relationship between diabetic nephropathy and an insertion (I)/deletion (D) polymorphism in intron 16 of the angiotensin-converting enzyme (ACE) gene is still under debate. The association of ACE gene polymorphism with nephropathy and retinopathy was therefore examined in 362 Japanese patients with non-insulin-dependent diabetes mellitus (NIDDM) and 105 healthy control subjects. Distribution of the ACE genotype did not differ between healthy control subjects and diabetic patients without complications. However, the frequency of the D allele was significantly higher in the diabetic subjects with nephropathy than in those without (0.32 in normoalbuminuric patients vs 0.44 in albuminuria patients with albuminuria) (χ2=7.7; p=0.006). There was no significant association between ACE genotype and retinopathy. These observations thus demonstrate a significant association of the ACE gene polymorphism with nephropathy, but not with retinopathy, in Japanese patients with NIDDM.

Polymorphism of the angiotensin-converting enzyme (ACE) gene in patients with thrombotic brain infarction.

The relationship between cerebrovascular disease and an insertion/deletion (I/D) polymorphism in intron 16 of the angiotensin-converting enzyme (ACE) gene is still being debated. We examined its role as a risk factor in patients with thrombotic brain infarction. The association between ACE polymorphism and ischemic stroke was examined in 181 patients with thrombotic brain infarction and 271 controls without strokes. The I/D polymorphism was examined using the polymerase chain reaction. Distributions of the ACE genotypes and alleles did not differ between the infarcted patients and the controls. Both distributions in patients with onset at age 60 years or younger were significantly higher than those in younger controls (genotype: chi 2 = 7.6, P = 0.02; allele: chi 2 = 5.6, P = 0.02). There were no significant differences in the distributions of ACE genotypes and alleles between the patients with lacunar infarcts and with cortical infarcts in all ages. There were also significant differences in the distribution of ACE genotypes and alleles between the younger and the elderly subgroup of patients with brain infarction (genotype: chi 2 = 12.9, P = 0.002; allele: chi 2 = 11.1, P = 0.0009). Furthermore, there was a significant decline in the frequency of the ACE D allele with increasing age in all patients with thrombotic brain infarction. These observations demonstrated a significant association between the ACE gene polymorphism and thrombotic brain infarction in patients age 60 years or younger in a Japanese population. Furthermore, there may be an association between the ACE D allele and mortality after cerebral infarction.

Characterization of self-glutamic acid decarboxylase 65-reactive CD4+ T-cell clones established from Japanese patients with insulin-dependent diabetes mellitus

To investigate autoimmunity to glutamic acid decarboxylase (GAD) 65 in Japanese patients with insulin-dependent diabetes mellitus (IDDM, type I diabetes), we established seven CD4+ T-cell clones, by stimulating peripheral blood mononuclear cells (PBMC) of six IDDM patients, using a mixture of overlapping human GAD65 peptides. No GAD65 autoreactive T-cell clones were evidenced in four healthy controls. Specificities of T-cell clones were as follows: (a) two clones specific to GAD65 p111-131 (residue 111 to 131) + DR53 (DRB4*0103); (b) one clone specific to GAD65 p413-433 + DR1 (DRB1*0101); (c) two clones specific to GAD65 p200-217 + either DR9 (DRB1*0901) or DR8 (DRB1*0802); and (d) two clones specific to GAD65 p368-388 + DP2 (DPA1*01 or 0201-DPB1*0201). Two DR53-restricted and one DR1-restricted T-cell clones, responded to a recombinant human GAD65 protein, and showed cytotoxicity against B lymphoblastoid cell lines pre-pulsed with the peptides. Six T-cell clones exhibited the Th1-like phenotype. Interestingly, two DR53-restricted T-cell clones killed a Fas-deficient B lymphoblastoid cell line, thereby indicating that cytotoxicity was not completely dependent on a Fas-Fas ligand interaction. Thus, the T-cell epitopes were mapped in a limited portion of GAD65 protein, with a tendency to be restricted by disease-associated HLA-DR, but not DQ molecules.

Renal complications in patients with diabetes mellitus associated with an A to G mutation of mitochondrial DNA at the 3243 position of leucine tRNA

The substitution of guanine for adenine at position 3243 of the leucine tRNA gene of mitochondrial DNA was originally described in association with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). Diabetes mellitus associated with the mutation (mitochondrial diabetes) is a different phenotype from MELAS. We identified 11 patients with the mutation among 385 Japanese diabetic patients: two had MELAS and nine had mitochondrial diabetes. We present data on a male patient with mitochondrial diabetes who developed the nephrotic syndrome at the age of 23. Light microscopy revealed mesangial expansion, PAS-positive deposits and segmental sclerosis in the glomeruli. Scattered mesangial electron-dense deposits and thickening of the basement membrane were found on electron microscopy, suggesting that diabetic glomerulosclerosis accompanied by focal glomerulosclerosis (FGS). Mitochondrial diabetes may pre-dispose patients to renal complications, including forms of glomerulonephritis, such as FGS.

Genetic control of immune response and disease susceptibility by the HLA-DQ gene.

The particular alleles of the HLA-DQ locus may control the low immune response to natural antigens by a dominant genetic trait through the immune suppression mediated by CD8+ suppressor T cells. The suppressor T cells may be activated by DQ-restricted and antigen-specific CD4+ suppressor/inducer T cells, because (1) a statistically significant association and linkage between low immune responsiveness to the natural antigens and the HLA-DQ gene were observed; (2) antigen-specific CD4+ T cells restricted by the DQ molecules encoded for by the HLA-DQ allele associated with low responsiveness were evidenced in many low responders; and (3) anti-HLA-DQ mAb restored the immune response to natural antigens, in some low responders. This HLA-DQ-controlled polymorphism of immune response to the natural antigens may account for the association between HLA-DQ alleles and organ-specific autoimmune diseases.

Circadian blood pressure variation in non-insulin-dependent diabetes mellitus with nephropathy

We studied a circadian blood pressure variation in relation to the progression of diabetic nephropathy in patients with non-insulin-dependent diabetes mellitus (NIDDM). Age, duration of diabetes, body mass index and glycemic control did not differ among the groups of patients with normo-, micro- and macroalbuminuria. None of the patients received antihypertensive drugs. There were no differences in renal and autonomic functions between normo- and microalbuminuric groups, but these functions were impaired in the macroalbuminuric group. The rise in blood pressure was more apparent in 24-h ambulatory blood pressure (AMBP), especially during night-time, as compared with casual blood pressure. Such blood pressure rise was in accordance with the progression of nephropathy. However, pulse rate did not differ among the three groups. The nocturnal fall in blood pressure was blunted in the micro- and macroalbuminuria groups, but evident in the normoalbuminuric group. In the latter, daytime systolic blood pressure (SBP) was significantly higher than night-time SBP (123 +/- 5 mmHg vs. 113 +/- 3 mmHg, P = 0.002). In contrast, in the former two groups of patients, there were no significant differences in SBP between daytime and night-time (134 +/- 9 mmHg vs. 134 +/- 9 mmHg, ns, for microalbuminuria and 159 +/- 8 mmHg vs. 165 +/- 7 mmHg, ns, for macroalbuminuria). Urinary albumin excretion was significantly correlated with night-time SBP (r = 0.48, P = 0.015), but not with daytime SBP (r = 0.30, ns).(ABSTRACT TRUNCATED AT 250 WORDS)

Allele-specific expression of the cytoplasmic exon of HLA-DQB1 gene

The β chain of the HLA-DQ molecule is shorter by eight amino acid residues than other major histocompatibility complex class II β chains due to elimination of the fifth exon coding for part of the cytoplasmic domain. This elimination is caused by one base substitution in the splice accepter site of the exon. We found that two HLA-DQB1 alleles, DQB1*0503 and DQB1*0601, did not have this substitution, and the exon was utilized in these two alleles. However, two forms of HLA-DQB mRNA, with or without exon 5, were generated in Epstein-Barr virus-transformed cell lines homozygous for DQB1*0503 or DQB1*0601, indicating alternative mRNA splicing. The alternative splicing of DQB1*0601 mRNA was also found in peripheral blood lymphocytes and L cell transfectants. To investigate the functional relevance of the allele-specific long cytoplasmic tail of HLA-DQ β chain, we developed three types of L cell transfectants expressing exclusively the HLA-DQw6 molecules with short cytoplasmic tail, long cytoplasmic tail, or both forms of the β chain, and used them as antigen presenting cells for streptococcal cell wall antigen-specific T cell lines. These three types of transfectants could function almost equally well as antigen presenting cells. It was thus demonstrated that both forms of HLA-DQ β chain, with or without eight amino acid residues coded for by the exon 5, can be associated with the HLA-DQ α chain, be expressed on the cell surface, and function as restriction molecules in antigen recognition by the CD4+ T cells.

Comparison of reliability of plasma fructosamine and glycosylated hemoglobin assays for assessing glycemic control in diabetic patients on hemodialysis

To search for a reliable marker of medium-term integrated blood glucose level in diabetics on maintenance hemodialysis (HD), plasma fructosamine and glycosylated hemoglobin (Hb) levels were determined every week and blood glucose levels were determined four times every day over 3 weeks. The mean values of fructosamine (mol/L per 40 g of albumin) and of glycosylated Hb of other the study period correlated (r = .746, P less than .001) for combined materials of diabetic and nondiabetic subjects. However, plasma fructosamine values at the end of the study period did not correlate with the overall mean blood glucose values during the preceding 8 to 21 days (r = .372, NS). In contrast, glycosylated Hb values correlated closely with the same mean blood glucose values (r = .703, P less than .001). Fructosamine values significantly decreased during a HD, irrespective of the increases in albumin and total protein. In conclusion, glycosylated Hb was a reliable marker of long-term integrated blood glucose even in diabetics on HD. However, fructosamine was not a reliable marker of medium-term integrated blood glucose in these patients.

Effect of gonadectomy on the development of diabetes mellitus, hypertension, and albuminuria in the rat model

Spontaneously hypertensive rats (SHR) given streptozotocin (STZ) neonatally developed genetic hypertension and overt hyperglycemia after the onset of puberty. In the present study, gonadectomy was performed before puberty in both males and females of this animal model. Orchidectomy suppressed the development of hypertension in vehicle-treated and STZ-treated SHR (systolic blood pressure at 11 weeks of age: 209 +/- 5 mm Hg in the intact vehicle group v 187 +/- 6 mm Hg in the orchidectomized vehicle group, P < .01; 211 +/- 14 mm Hg in the intact STZ group v 182 +/- 4 mm Hg in the orchidectomized STZ group, P < .001). Furthermore, orchidectomy ameliorated the development of overt hyperglycemia in STZ-treated SHR (nonfasting plasma glucose at 12 weeks of age: 22.1 +/- 0.7 mmol/L in the intact group v 16.1 +/- 2.4 mmol/L in the orchidectomized group, P < .05). On the other hand, orchidectomy did not affect glucose tolerance in vehicle-treated SHR, but attenuated the insulin response to an oral glucose load (P < .05). Orchidectomy significantly decreased urinary albumin excretion and kidney weight in both the vehicle and the STZ groups. Ovariectomy significantly increased body weight gain irrespective of STZ treatment. However, ovariectomy had no effect on hypertension, hyperglycemia, albuminuria, or kidney weight in either vehicle or STZ groups. This study demonstrated that gonadectomy had protective effects against development of hypertension, hyperglycemia, and albuminuria in males but not in females. This suggests that sex hormones may be important as a link between diabetes mellitus and hypertension in males.

Renoprotective effect of enalapril in uninephrectomized spontaneously hypertensive rats with neonatal streptozotocin-induced diabetes

We compared the renoprotective effect between angiotensin-converting enzyme inhibitor, enalapril, and a dihydropyridine-type calcium channel blocker, nicardipine, in a severe form of renal injury in rats. Two-day-old spontaneously hypertensive rats (SHR) were injected with streptozotocin or vehicle as control. UNX was performed at 3 weeks of age, and enalapril or nicardipine was administered in drinking water from 7 weeks of age. Uninephrectomy (UNX) markedly exacerbated hypertension and renal injury in the nondiabetic and diabetic SHR. Enalapril and nicardipine comparably reduced blood pressure in UNX diabetic SHR. However, serum creatinine was significantly elevated in the nicardipine-treated group as compared with the enalapril-treated group at 24 weeks of age (nicardipine-treated group, 67 +/- 4 microM; enalapril-treated group, 49 +/- 3 microM; P < 0.01; untreated group 57 +/- 4 microM). Furthermore, the incidence of glomerular sclerosis was similar between untreated and nicardipine-treated groups, whereas it tended to be reduced in the enalapril-treated group. In a separate experiment of diabetic SHR without UNX, enalapril therapy significantly ameliorated hyperglycemia and albuminuria (P < 0.01). This study showed that a renoprotective effect was seen in enalapril but not in nicardipine in UNX diabetic SHR despite the comparable reduction of blood pressure. This suggests that enalapril may be more effective than nicardipine in delaying the progression of a severe form of diabetic nephropathy.