Toshihide Shike

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.

Susceptibility and negative epistatic loci contributing to type 2 diabetes and related phenotypes in a KK/Ta mouse model

The KK/Ta mouse strain serves as a suitable polygenic model for human type 2 diabetes. Using 93 microsatellite markers in 208 KK/Ta x (BALB/c x KK/Ta)F1 male backcross mice, we carried out a genome-wide linkage analysis of KK/Ta alleles contributing to type 2 diabetes and related phenotypes, such as obesity and dyslipidemia. We identified three major chromosomal intervals significantly contributing to impaired glucose metabolism: one quantitative trait locus for impaired glucose tolerance on chromosome 6 and two loci for fasting blood glucose levels on chromosomes 12 and 15. The latter two loci appeared to act in a complementary fashion. Two intervals showed significant linkages for serum triglyceride levels, one on chromosome 4 and the other on chromosome 8. The KK allele on chromosome 8 acts to promote serum triglyceride levels, whereas the KK allele on chromosome 4 acts to suppress this effect in a recessive fashion. In addition, it is suggested that the chromosome 4 locus also acts to downregulate body weight and that the chromosome 8 locus acts to upregulate serum insulin levels. Our data clearly showed that each disease phenotype of type 2 diabetes and related disorders in KK/Ta mice is under the control of separate genetic mechanisms. However, there appear to be common genes contributing to different disease phenotypes. There are potentially important candidate genes that may be relevant to the disease.

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.

Genetic susceptibility to type 2 diabetic nephropathy in human and animal models

SUMMARY:  Diabetic nephropathy is the most common cause of end‐stage renal disease (ESRD) in Japan, Western Europe, and the United States. Mega studies such as Diabetes Control and Complication Trial (DCCT), Epidemiology of Diabetes Interventions and Complications (EDIC), and the United Kingdom Prospective Diabetes Study (UKPDS) clarified that poor glycemic and blood pressure control are undoubtedly involved in the development of nephropathy. However, these factors are not sufficient to predict which diabetic patients will develop renal disease, because not all patients with poor glycemic and blood pressure control develop renal disease. Since ethnic variations and familial clustering of diabetic nephropathy have been observed, genetic factors might contribute to susceptibility to this disease. Several methods such as (genome wide) association studies, sib‐pair analysis, and quantitative trait loci (QTLs) analysis are available to examine polygenic diseases. However, no mutations that could explain the majority of nephropathy cases have been identified so far. The development of most diabetic nephropathy might be explained by the polygenic effect (i.e. many minor gene‐gene interactions might be very important in the development of nephropathy). Identification of candidate genes of nephropathy enables targeting of therapy in patients at risk and development of novel therapeutic agents.

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.

Effect of calcium channel blockers, nifedipine and benidipine, on death of cultured mouse mesangi al cells.

We examined the effects of the short–acting calcium channel blocker (CCB) nifedipine and the long–acting CCB benidipine on the death of mouse cultured mesangial cells induced by tumor necrosis factor alpha (TNF–α) and/or cycloheximide (CHX). Cell death was evaluated by a morphological study using semithin sections. The dead cells were divided into three types, i.e., apoptotic cells (type 1), necrotic cells (type 3) and other types of dead cells, the so–called ‘secondary necrotic cells‘ or ‘postapoptotic necrotic cells’ (type 2). In the morphological study with semithin sections, cells in the presence of TNF–α or CHX and nifedipine or benidipine showed low percentages of all dead cell types with 24 h incubation. Both nifedipine and benidipine have protective effects against TNF–α or CHX. It is postulated that CCB might inhibit the apoptotic or necrotic processes by TNF–α or CHX with 24 h incubation. With 36 h incubation, CCB increased the percentages of all types of dead cells except for treatment with 1×10–5 M benidipine and CHX. It appears that these cell–protective effects might be decreased after treatment with TNF–α or CHX and CCB for 36 h. In conclusion, the short–acting CCB nifedipine and the long–acting CCB benidipine have protective effects on mouse cultured mesangial cells against TNF–α or CHX . However, nifedipine and benidipine did not inhibit specific types of cell death using semithin sections in this study.