Hideyuki Kurioka

Increased Excretion of Urinary Transforming Growth Factor Beta 1 in Patients with Diabetic Nephropathy

The accumulation of extracellular matrix in the glomeruli of human and experimental models of diabetic nephropathy is associated with disease progression. Transforming growth factor beta 1 (TGF-β1), which is a multifunctional peptide growth factor, plays a key role in the synthesis of extracellular matrix protein in vitro, and the expression of TGF-β1 is elevated in human and rat diabetic nephropathy. In this study, we measured the urinary TGF-β1 excretion in 57 patients with non-insulin-dependent diabetes mellitus and in 20 healthy volunteers to examine whether the determination of urinary TGF-β1 excretion would facilitate the evaluation of the degree of mesangial expansion in patients with diabetic nephropathy. Both active and total TGF-β1 levels in 24-hour urine samples collected from patients with diabetes mellitus and normal controls were measured using an enzyme-linked immunosorbent assay. We observed a higher excretion of urinary TGF-β1 in patients with diabetes mellitus than in normal controls. In addition, the urinary TGF-β1 excretion was elevated in patients with severe mesangial expansion. These results suggest that urinary TGF-β1 may represent one parameter that can be used to evaluate the progression of diabetic nephropathy.

Cell proliferation and apoptosis of the glomerular epithelial cells in rats with puromycin aminonucleoside nephrosis.

Injury and repair of the glomerular epithelial cells (GECs) play an important role in the pathogenesis of focal segmental glomerulosclerosis (FSGS). To obtain a better understanding of proliferation and apoptosis of GECs, we examined immunohistochemical and in situ hybridization findings in puromycin aminonucleoside nephrosis (PAN) of rats. The minimal-change nephrotic syndrome model (PAN-MCNS) was induced by administering 5 subcutaneous injections of puromycin aminonucleoside (PA; each 1.5 mg/100 g B/W to one group of rats), whereas the FSGS model (PAN-FSGS) was induced by administering an additional 5 injections of PA to another group of rats. The cell kinetics of the GECs were assessed with labeling 5-bromo 2′-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA). To investigate regulation of apoptosis in rats with PAN, we evaluated the expression of p53, Fas antigen, Fas ligand and Bcl-2. Rats with PAN-MCNS exhibited a significantly greater number of BrdU- and PCNA-labeled GECs as compared with control rats. In rats with PAN-FSGS, the number of PCNA-labeled GECs was greater than in rats with PAN-MCNS, but the number of BrdU-labeled GECs was lower. Apoptotic cells were occasionally observed in the sclerotic lesions, with the number being significantly higher in rats with PAN-FSGS than in rats with PAN-MCNS and control. Apoptotic cells were observed in the GECs of PAN-FSGS rats. However, they were negative for p53, Fas antigen, and Fas ligand. Immunohistochemical and in situ hybridization studies revealed a greater intraglomerular overexpression of Bcl-2 protein and bcl-2 mRNA in the PAN-FSGS rats as compared with control rats. These results suggest that insufficient proliferation and apoptosis in GECs may be involved in the progression of FSGS.

Plasma and Urinary Levels of Adrenomedullin in Chronic Glomerulonephritis Patients with Proteinuria

In this study, we measured levels of plasma and urinary adrenomedullin (AM) in 37 patients with chronic glomerulonephritis including minimal change nephrotic syndrome, focal segmental glomerulosclerosis or membranous nephropathy that can induce severe proteinuria. Thirty-nine healthy volunteers were enrolled as controls. Plasma and urinary AM levels were measured by an AM-specific radioimmunoassay. Plasma AM concentrations were higher and urinary AM levels were lower in patients with chronic glomerulonephritis than in healthy volunteers. Patients were divided into two groups according to urinary excretion of protein for 24 h (UPro, g/day) which reflects the disease activity or glomerular damage of the glomerulonephritis (group I: Upro < 1, group II: Upro ≥ 1). Plasma AM levels positively and urinary AM-levels negatively correlated with the degree of proteinuria. These results suggest that plasma and urinary AM levels in patients with chronic glomerulonephritis reflect the disease activity or glomerular damage represented by the degree of proteinuria.

Adrenomedullin Gene Transcription Is Decreased in Peripheral Blood Mononuclear Cells of Patients with IgA Nephropathy

We measured mRNA levels of adrenomedullin (AM), C-type natriuretic peptide (CNP), vascular endothelial growth factor (VEGF), interleukin 1β (IL-1β) and interleukin 6 (IL-6) in peripheral blood mononuclear cells (PBMC) of patients with IgA nephropathy. To evaluate these mRNA levels, we employed a real-time quantitative PCR method which was performed using a hybridization probe labeled with two fluorescence dyes. This strategy was found to afford the standard curves with a high correlation, suggesting that this method is useful for evaluations of mRNA levels. By this method, levels of AM, CNP, VEGF, IL-1β and IL-6 mRNA in PBMC of 49 IgA nephropathy patients and 35 healthy volunteers were evaluated. Among the mRNAs examined, AM mRNA levels were significantly lower in severe-grade than in mild-grade IgA nephropathy patients. Furthermore, AM mRNA levels correlated with CNP mRNA levels in PBMC of patients with IgA nephropathy, and each peptide generated from these mRNAs has antiproliferative effects on mesangial cells. These data indicate that gene expression of AM in PBMC is regulated according to the pathophysiological states of IgA nephropathy and that decreased AM production may contribute to the progression of IgA nephropathy.

Association of Interleukin-1 Receptor Antagonist Gene Polymorphism with IgA Nephropathy

It is evident that cytokines play an important role in the pathogenesis as well as disease progression of IgA nephropathy (IgAN). Several gene polymorphisms of the pertinent cytokines have an influence on the level of cytokine production. Interleukin-1 receptor antagonist (IL-1ra) gene polymorphism has been found to affect disease susceptibility and activity in several inflammatory diseases. In the present study, we analyzed polymorphism of the variable number tandem repeat (VNTR) of IL-1ra in patients diagnosed as having IgAN (n = 106) and normal controls (n = 74). The allele frequency of IL-1ra polymorphism in IgAN patients was not statistically different from that of the control group. There was no significant difference in the carriage rate of the two-repeat allele (IL1RN*2) between IgAN patients and the control group (8.5 vs. 6.8%). The carriage rate of IL1RN*2 was significantly higher in IgAN patients with severe proteinuria (≧1.6 g/day) or increased serum creatinine level (≧2.0 mg/dl; p < 0.05). Furthermore, the carriage rate of IL1RN*2 was significantly higher in patients with severe mesangial cell proliferation (p < 0.01). Our results suggest that IL-1ra polymorphisms are not associated with the development of IgAN in Japanese patients but the presence of IL1RN*2 may be associated with increased disease activity.

Increased urinary levels of adrenomedullin in patients with cystitis

In this study, we examined urinary levels of adrenomedullin (AM) in 18 healthy volunteers and 18 patients with cystitis. We also compared urinary levels of AM in 11 patients with cystitis before and after antibiotic treatment. Urinary AM concentrations were measured by a radioimmunoassay specific for human AM. Urinary AM levels in patients with cystitis were significantly elevated compared with those of healthy volunteers and correlated positively with the number of urine leukocytes. By antibiotic treatment, urinary AM levels significantly decreased as compared with before the treatment. By RNA blot analysis of AM transcript, we detected significant levels of AM mRNA in canine urinary bladder and ureter. Intravenous administration of lipopolysaccharide elevated the AM mRNA level in the urinary bladder. These data suggest that infection and inflammation stimulate AM production in the urinary tract, which results in increased urinary AM levels in patients with cystitis. Based on these results, it is deduced that AM participates in the pathophysiology of cystitis, and its urinary level could be used as an index of the degree of cystitis.

Vascular endothelial growth factor mRNA synthesis by peripheral blood mononuclear cells in patients with acute myocardial infarction

We studied vascular endothelial growth factor (VEGF) mRNA synthesis by peripheral blood mononuclear cells (PBMCs) in 30 patients with acute myocardial infarction (AMI) and 20 healthy individuals. PBMCs were isolated from all patients on days 3 and 14 after the onset of aMI, and from all of control individuals. To prepare samples containing identical amounts of GAPDH cDNA, competitive PCR was performed by co-amplifying serial dilutions of GAPDH mutant templates. Next, to measure VEGF cDNA quantitatively in the samples containing identical amounts of GAPDH, we also used competitive PCR by co-amplifying mutant templates of VEGF. The serum VEGF concentrations on day 14 in patients with aMI were measured by an ELISA method. Higher levels of VEGF mRNA in PBMCs were present on day 14 than either on day 3 or in the control group. Serum VEGF concentrations correlated with the VEGF mRNA levels of PBMCs on day 14. Peak serum CK levels correlated well with VEGF mRNA levels of PBMCs on day 14. The present findings suggest that PBMCs may be one of the candidates responsible for elevated circulatory VEGF protein following aMI. In addition, VEGF mRNA may be overexpressed in PBMCs in response to cardiac muscle damage.

Gene polymorphism and clinical manifestations in Japanese patients with IgA nephropathy

To investigate the influence of genetic polymorphism on clinical manifestations of IgA nephropathy (IgAN), we studied the gene polymorphism of angiotensin-I converting enzyme (ACE), angiotensinogen (Ang), angiotensin II type 1 receptor (AngT1R), and interleukin-1 receptor antagonist (IL-1Ra) in Japanese patients with IgAN. A total of 85 patients (29 males and 56 females) with IgAN and 100 healthy controls were enrolled in this study. All patients gave informed consent to participate in this study. The average follow-up period in this study was 5.5 years. Gene polymorphism was determined by the polymerase chain reaction (PCR) method. Twenty four-hour urinary protein excretion and serum creatinine were measured by routine chemical methods. Renal biopsy specimens were examined before treatment in all patients to evaluate the degree of mesangial proliferation. There were no differences in either the genotype or allele frequency of the D/I polymorphism between the patients with IgAN and normal controls (D allele frequency; 39 and 40%, respectively). In patients with worsening renal function versus those without progression in follow-up period, ACE genotype frequencies were 20, 40 and 40% versus 17, 46 and 37%, for DD, DI, and II genotypes, respectively. The levels of urinary protein excretion were not different among these three genotypes. Furthermore, there were no significant associations between ACE polymorphism and histological findings in patients with IgAN. The deviation of Ang M235T and Ang T174M gene polymorphism in patients with IgAN did not differ from those of normal controls (36, 47 and 17% vs 34, 50, and 16% for MM, MT, and TT genotypes, respectively, in Ang M235T polymorphism, 6, 11 and 83% vs 4, 15 and 81%, respectively, for TT, TM, and MM genotypes, respectively, in Ang T174M polymorphism). The frequency in patients who excreted over 1 g/day proteinuria was 10, 23 and 28% for MM, MT, and TT genotypes, respectively, in Ang M235T polymorphism (P < 0.05). There were no significant associations between Ang T174M polymorphism and the clinical features of IgAN. There were no differences in the genotype frequencies of the AngT1R polymorphism between the patients with IgAN and normal controls (81, 17 and 2% vs 88, 12 and 0% for AA, AC, and CC genotypes, respectively). AngT1R polymorphism was not associated with the levels of urinary protein excretion, the degree of mesangial proliferation, and deterioration of renal function. The allele frequency of the IL-1Ra polymorphism was not different between IgA nephropathy and normal controls (94, 4 and 2% vs 96, 3 and 1% for 4-, 2-, and 1repeat allele, respectively), but the degree of mesangial proliferation was severe in patients with two-repeat allele in IL-1Ra polymorphism (56, 11 and 33% vs 10, 45 and 45% for severe, moderate, and mild mesangial proliferation, respectively, in patients with two-repeat allele vs those without two-repeat allele in IL-1Ra polymorphism). IL-1Ra polymorphism was not associated with the levels of urinary protein excretion and deterioration of renal function. Ang M235T and IL-1Ra polymorphism may influence the progression of renal function in Japanese patients with IgAN. ACE, Ang T174M, and AngT1R polymorphism are not associated with the clinical manifestations of IgA nephropathy. Gene polymorphism and clinical manifestations in Japanese patients with IgA nephropathy