Yuichiro Makita

Relationship between polymorphism in the angiotensinogen, angiotensin-converting enzyme or angiotensin II receptor and renal progression in Japanese NIDDM patients.

We determined the relationship between the gene polymorphism of angiotensinogen (AGT), angiotensin-converting enzyme (ACE), or angiotensin II receptor (AT1R) and the progression of diabetic nephropathy in a multicenter trial of ethnically homogeneous Japanese patients with non-insulin-dependent diabetes (NIDDM). Gene polymorphism of ACE I/D, AGT M235T and AT1R A1166C was determined by polymerase chain reaction amplification using allele-specific primers. Japanese NIDDM patients (n = 1,152) were selected from several diabetic clinics. All patients were divided into three groups as follows: (1) group I (n = 407): normoalbuminuric patients; (2) group II (n = 327): microalbuminuric patients, and (3) group III (n = 418): overt albuminuric patients. Clinical factors for investigation in all patients were the date of birth, gender, levels of urinary albumin excretion, findings of the ocular fundus, duration of diabetes, hemoglobin A1c and blood pressure. It appears that genetic polymorphisms in the renin-angiotensin systems, i.e. ACE or AT1R, may affect the progression to renal failure of patients (especially females) with NIDDM.

High-density SNP genome wide linkage scan for susceptibility genes for diabetic nephropathy in type 1 diabetes: Discordant sib-pair approach.

OBJECTIVE— Epidemiological and family studies have demonstrated that susceptibility genes play an important role in the etiology of diabetic nephropathy, defined as persistent proteinuria or end-stage renal disease (ESRD) in type 1 diabetes. RESEARCH DESIGN AND METHODS— To efficiently search for genomic regions harboring diabetic nephropathy genes, we conducted a scan using 5,382 informative single nucleotide polymorphisms on 100 sibpairs concordant for type 1 diabetes but discordant for diabetic nephropathy. In addition to being powerful for detecting linkage to diabetic nephropathy, this design allows linkage analysis on type 1 diabetes via traditional affected sibpair (ASP) analysis. In weighing the evidence for linkage, we considered maximum logarithm of odds score (maximum likelihood score [MLS]) values and corresponding allelic sharing patterns, calculated and viewed graphically using the software package SPLAT. RESULTS— Our primary finding for diabetic nephropathy, broadly defined, is on chromosome 19q (MLS = 3.1), and a secondary peak exists on chromosome 2q (MLS = 2.1). Stratification of discordant sibpairs based on whether disease had progressed to ESRD suggested four tertiary peaks on chromosome 1q (ESRD only), chromosome 20p (proteinuria only), and chromosome 3q (two loci 58 cm apart, one for ESRD only and another for proteinuria only). Additionally, analysis of 130 ASPs for type 1 diabetes confirmed the linkage to the HLA region on chromosome 6p (MLS = 9.2) and IDDM15 on chromosome 6q (MLS = 3.1). CONCLUSIONS— This study identified several novel loci as candidates for diabetic nephropathy, none of which appear to be the sole genetic determinant of diabetic nephropathy in type 1 diabetes. In addition, this study confirms two previously reported type 1 diabetes loci.

Detection of cell death of cultured mouse mesangial cells induced by oxidized low-density lipoprotein

The objectives of the present study using cultured mouse mesangial cells (MMC) were (1) to evaluate the type of cytotoxicity induced by oxidized (ox) LDL, i.e. apoptosis, necrosis and types of other cell death and (2) to investigate the pathway of cell death under incubation with antioxidants or scavenger receptor (SR) antagonists. LDH release and a morphological examination were used in this study. Trypan blue staining of MMC was performed to detect dead cells in culture. Cytotoxicity of ox-LDL in MMC was found to be dose- and time-dependent. In the morphological study of electron microscopy, three different types of cell death in ox-LDL-treated MMC were identified. In the morphological study with semithin sections, these three types of dead cells were identified at different dosages of ox-LDL. Type 1 or type 2 dead cells were observed in low dose ox-LDL or in middle-dose ox-LDL-treated MMC, respectively. Type 3 dead cells were marked in high dose ox-LDL-treated MMC. It appears that the cells were apoptotic (type 1), necrotic (type 3) and other types (type 2). The cytotoxicity of ox-LDL was not mediated by cellular internalization of ox-LDL via SRs. On the other hand, the cytotoxicity of ox-LDL was inhibited by antioxidants such as α-tocopherol, probucol, N-acetyl-cysteine or glutathione ethyl ester. It is indicated that the pathways of ox-LDL induced cell death were distinct from the pathway via SRs.

Increase of Urinary Type IV Collagen in Normoalbuminuric Patients with Impaired Glucose Tolerance

Accessible online at: http://BioMedNet.com/karger Dear Sir, Microalbuminuria is the only early clinical sign of the subsequent diabetic nephropathy. However, it is also known that significant structural changes have already appeared even at the stage of microalbuminuria in non-insulin-dependent diabetes mellitus (NIDDM) patients. Thus, it is necessary to develop a more sensitive measurement for detecting the early stage of renal injury in patients with diabetic nephropathy. Since type IV collagen is the principal component of glomerular basement membrane and mesangial matrix, the levels of type IV collagen in sera and urinary samples may reflect the rate of its turnover, such as the balance of production by intrinsic renal cells and degradation by matrix proteinases in diseased kidneys. To investigate the alteration of renal turnover of type IV collagen in patients with impaired glucose tolerance (IGT), urinary type IV collagen (uIV) was measured by a highly sensitive one-step sandwich enzyme immunoassay (EIA) (Fuji Chemical Industries, Co. Ltd, Takaoka, Toyama, Japan) [1]. Diagnosis of NIDDM was made according to the criteria of the 75-gram oral glucose tol-

Dissociated Expression of Collagen Type IV Subchains in Diabetic Kidneys of KKAy Mice

Diabetic nephropathy is characterized by thickening of the glomerular basement membrane and expansion of the mesangial matrix. The glomerular basement membrane is assembled from at least five genetically distinct collagen IV chains. In patients with diabetic nephropathy, differential distribution of these components has been demonstrated. In order to clarify the relationship between progression of diabetic nephropathy and altered type IV collagen assembly in the renal cortex, we examined steady state mRNA levels encoding collagen IV subchains in the kidney cortices of spontaneously diabetic KKAy mice and nondiabetic C57black mice as controls. They were sacrificed at 4, 8, 16, and 24 weeks of age. Northern and dot blot analyses were performed using 32P-labeled mouse probes for classical α1(IV) and α2(IV) and for α3(IV), α4(IV), and α5(IV) minor chains. The mRNA levels for all collagen IV chains peaked at 4 weeks of age and declined rapidly thereafter in the nondiabetic mice. At all times, α1(IV) and α2(IV) mRNA expressions were abundant and almost unchanged in KKAy mice. In contrast, mRNA levels for α3(IV), α4(IV), and α5(IV) progressively changed with age. It appears that the expression of minor collagen IV chains is dissociated from the α1(IV) and α2(IV) chains in diabetic nephropathy. Moreover, an unbalanced increase in the production may affect collagen IV assembly and contribute to basement membrane thickening in diabetic nephropathy of KKAy mice.

Expression of Cysteine Proteinases and Their Inhibitor, Cystatin β, in Cultured Rat Mesangial Cells

Matrix expansion in the glomerular mesangial area is observed in diabetic nephropathy. Intracellular breakdown of long-lived proteins was lower in mesangial cells in the high glucose medium than that in the control medium. Enzymatic activity of cathepsin L increased 1.4-fold after 6 h of treatment with the high glucose, and then declined gradually to 72% of control cells after treatment for 36 h. Change in the enzyme activity of cathepsin B showed a similar time course but less magnitude than that of cathepsin L. Immunoblot analysis with anti-cathepsin L antibody showed that change in the enzyme activity of cathepsin L was due to the change in the amount of cathepsin L, and that with anti-cathepsin B antibody showed no change in the amount of cathepsin B in the mesangial cells treated with high glucose. Intracellular cathepsin activities were controlled not only by the amounts but also by the inhibitor cystatin beta. Immunoblot analysis with anti-cystatin beta antibody showed that intracellular levels of cystatin beta increased slightly after 24 h of treatment with high glucose. These changes were derived from changes in mRNA level. These results, therefore, demonstrated that the decrease of intracellular protein breakdown in mesangial cells treated with high glucose medium was due to both suppression of cathepsins and increase of cystatin beta.

Pathogenesis and Treatment of Type 2 Diabetic Nephropathy: Lessons from the Spontaneous KK/Ta Mouse Model

Diabetic nephropathy is a major cause of end-stage renal failure (ESRF) in patients with both type 1 and type 2 diabetes. Many factors such as genetic and non-genetic promoters, hypertension, hyperglycemia, accumulation of advanced glycation end products (AGEs), dyslipidemia, albuminuria and proteinuria influence the progression of this disease. It is important to determine pathogenesis and treatment of this disease. However, it is difficult to investigate since human diabetes is a heterogeneous and multifactorial disease. Therefore, most of these mechanisms have been investigated in animal experiments. KK/Ta mice have a clearly different genetic background in terms of body weight, blood glucose, impaired glucose tolerance (IGT), urinary albumin excretion and serum triglyceride than BALB/c mice. Renal lesions of KK/Ta mice closely resemble those in human early diabetic nephropathy. Thus, the KK/Ta mouse may serve as a suitable model for the study of type 2 diabetes and early diabetic nephropathy in humans. We reviewed genetic susceptibility using genome-wide linkage analysis and differential display polymerase chain reaction (DD-PCR) or Northern blot analysis, and treatment of diabetic nephropathy using angiotensin type 1 (AT1) receptor blockers (ARB) or thiazolidinediones (TZDs) in KK/Ta mice.

Dilazep Hydrochloride, an Antiplatelet Drug, Inhibits Lipopolysaccharide-Induced Mouse Mesangial Cell IL-6 Secretion and Proliferation

Background: Antiplatelet agents have been widely used to reduce proteinuria and to prevent the progression of chronic glomerulonephritis or diabetic nephropathy to end-stage renal failure. Dipyridamole, one type of antiplatelet drug, inhibits the proliferation of glomerular mesangial cells (MCs). The effect of dilazep hydrochloride (dilazep) on these cells is still obscure. The effects of dilazep on cultured MC IL-6 secretion and proliferation were investigated in the present study. Methods: IL-6 secretion from MC induced by bacterial lipopolysaccharide (LPS) were assessed using sandwich ELISA. LPS-induced MC proliferation was detected by 3H-thymidine incorporation and WST-1 assay (similar to MTT assay). Results: Incubation of MCs with various dosages of LPS (0, 1, 10, 50 and 100 ng/ml) induced IL-6 secretion in a dose-dependent manner. However, dilazep significantly inhibited this LPS-induced IL-6 secretion from MCs in a dose- and time-dependent manner. Dilazep also significantly inhibited MC proliferation in a dose-dependent manner. Conclusion: It appears that these effects of dilazep may prevent progression of mesangial proliferative glomerulonephritis.

Effects of Enalapril Treatment on Gene Expression of Smooth Muscle Myosin Heavy Chain Isoforms in Glomeruli of Diabetic Rats

We have previously shown that smooth muscle contains three types of myosin heavy chains: SM1, SM2, and SMemb. The present study was designed to assess how glomerular expression of mRNA for these isoforms is regulated and whether their expression is affected by enalapril treatment in diabetic rats. Animals were divided into 4 groups: (1) untreated diabetic rats; (2) enalapril-treated diabetic rats; (3) untreated control rats, and (4) enalapril-treated control rats. Enalapril treatment was continued for 24 weeks. The glomerular mRNA levels for SM1 and SM2 showed little change in all groups throughout the experimental period. In contrast, SMemb mRNA in group 1 increased significantly with age compared to levels found in untreated controls (4.6-fold higher at 4 weeks, p < 0.01; 6.8-fold higher at 12 weeks, p < 0.01, and 10.6-fold higher at 24 weeks, p < 0.001). Enalapril reduced both creatinine clearance (p < 0.01) and urinary protein excretion (p < 0.01) in diabetic rats. Moreover, enalapril significantly attenuated the increase in the glomerular SMemb mRNA level in diabetic rats (the difference between treated and untreated rats was significant at p < 0.01 from week 4 to 24). However, enalapril had no effect on SMemb mRNA levels in controls. These data suggest that SMemb is a molecular marker for phenotypic alteration and that the beneficial effect of enalapril on proteinuria and renal function may be, at least in part, associated with reducing SMemb mRNA expression in diabetic glomeruli.

Effect of Beraprost Sodium, a PGI2 Analogue, on Proliferation of Cultured Rat Glomerular Mesangial Cells

Dr. Yasuhiko Tomino, Division of Nephrology, Department of Medicine, Juntendo University School of Medicine, Tokyo 113 (Japan) Dear Sir, A study of the effect of beraprost sodium (BPS) on proliferation of cultured rat glomerular mesangial cells is described. Prosta-cyclin (PGI2) synthesized from arachidonic acid by cyclo-oxygenase is a potent relaxant of vascular smooth muscle and a anti-aggre-gatory agent [1]. BPS, a stable analogue of PGI2, has a long biological half-life and is orally effective in inhibiting the aggregation of platelets [2]. Recently, Wang et al. [3] reported that BPS might improve albumin-uria and glomerular filtration rate (GFR) due to its vasodilating effects in the early phase of streptozotocin (STZ)-induced diabetic rats. Sakai et al. [4] examined the cyto-protective effect of BPS in cultured human endothelial cells from the umbilical vein. They showed an effect of BPS on proliferation of endothelial cells and speculated contribution to an increase in cyclic AMP (cAMP) level in endothelial cells treated with BPS. Davison et al. [5] reported a pro-liferative effect of dibutyryl cAMP in human dermal microvessel endothelial cells, but not in human umbilical cord vein endothelial cells. This effect appeared at a concentration of 5 × 10-4M, but at not more than 1 × 10-3Af or less than 1 × 10-4M [5]. However, the effect of BPS on glomerular mesangial cells has remained unclear. In this study, rat glomerular mesangial cells were cultured with 0.5% fetal calf serum (FCS) in RPMI 1640 for 48 h. The cells were incubated with 200 mg/dl or 500 mg/dl of glucose and 0-30 μmol/l of BPS in RPMI 1640 containing 10% FCS for 18 h. Thereafter, proliferation of the glomerular mesangial cells was determined by the 5-bromo-2’-deoxy-uridine labelling and detection kit III. Proliferation of glomerular mesangial cells was suppressed after incubation with highglucose medium without BPS treatment. In high-glucose medium, the proliferation of the mesangial cells under the incubation with low dose BPS (0.1-0.3 μg/l) was significantly greater than that without BPS (p < 0.05). On the other hand, the proliferation of the mesangial cells under incubation with high-dose BPS (10-30 μg/l) was significantly lower than that without BPS in the same medium (p < 0.05). These findings suggest that the low-dose BPS might improve the suppression of mesangial cell proliferation in the high-glucose medium. It