Tomoya Umezono

A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes

It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A carboxylase beta (ACACB) as a candidate for a susceptibility to diabetic nephropathy; the landmark SNP was found in the intron 18 of ACACB (rs2268388: intron 18 +4139 C > T, p = 1.4×10−6, odds ratio = 1.61, 95% confidence interval [CI]: 1.33–1.96). The association of this SNP with diabetic nephropathy was examined in 9 independent studies (4 from Japan including the original study, one Singaporean, one Korean, and two European) with type 2 diabetes. One case-control study involving European patients with type 1 diabetes was included. The frequency of the T allele for SNP rs2268388 was consistently higher among patients with type 2 diabetes and proteinuria. A meta-analysis revealed that rs2268388 was significantly associated with proteinuria in Japanese patients with type 2 diabetes (p = 5.35×10−8, odds ratio = 1.61, 95% Cl: 1.35–1.91). Rs2268388 was also associated with type 2 diabetes–associated end-stage renal disease (ESRD) in European Americans (p = 6×10−4, odds ratio = 1.61, 95% Cl: 1.22–2.13). Significant association was not detected between this SNP and nephropathy in those with type 1 diabetes. A subsequent in vitro functional analysis revealed that a 29-bp DNA fragment, including rs2268388, had significant enhancer activity in cultured human renal proximal tubular epithelial cells. Fragments corresponding to the disease susceptibility allele (T) had higher enhancer activity than those of the major allele. These results suggest that ACACB is a strong candidate for conferring susceptibility for proteinuria in patients with type 2 diabetes.

Replication Study for the Association Between Four Loci Identified by a Genome-Wide Association Study on European American Subjects With Type 1 Diabetes and Susceptibility to Diabetic Nephropathy in Japanese Subjects With Type 2 Diabetes

OBJECTIVE Genetic factors are believed to contribute to the development and progression of diabetic nephropathy. Recently, a genome-wide association study for diabetic nephropathy revealed four novel candidate loci in European American subjects with type 1 diabetes. In this study, we determined the association of the four loci with diabetic nephropathy in Japanese subjects with type 2 diabetes. RESEARCH DESIGN AND METHODS We genotyped 11 singlenucleotide polymorphisms (SNPs) in four distinct loci (rs39059 and rs39075 in the CPVL/CHN2, rs1888747 and rs10868025 in FRMD3, rs739401 and rs451041 in CARS, and rs1041466, rs1411766, rs6492208, rs7989848, and rs9521445 in a chromosome 13q locus) in four independent Japanese populations. RESULTS Six SNPs were nominally associated with diabetic nephropathy in one of the four Japanese populations (P < 0.05; rs451041 in study 1; rs39059 and rs1888747 in study 3; rs1411766 in studies 1 and 4; and rs7989848 and rs9521445 in study 4); however, no significant association was observed for any SNP after correction for multiple testing errors in the individual populations. Nevertheless, a meta-analysis performed for the data obtained from all four populations revealed that one SNP (rs1411766) in chromosome 13q was significantly associated with diabetic nephropathy in the Japanese populations (nominal P = 0.004, corrected P = 0.04, odds ratio 1.26 [95% CI = 1.07–1.47]). CONCLUSIONS Our results suggest that the rs1411766 locus may be commonly involved in conferring susceptibility to diabetic nephropathy among subjects with type 1 or type 2 diabetes across different ethnic groups.

Glomerular expression of platelet-derived growth factor (PDGF)-A, -B chain and PDGF receptor-Α, -Β in human diabetic nephropathy

BackgroundMesangial expansion is thought to be a major cause of diabetic nephropathy (DN). Platelet-derived growth factor (PDGF) plays an important role in the production of extracellular matrix proteins in renal diseases. The present study was designed to determine the expression of PDGF and PDGF receptor (PDGFR) mRNA in the renal tissues of type 2 diabetic patients with DN.MethodsWe examined open renal biopsies of 20 type 2 diabetic patients with DN, and 10 normal human kidneys (NHK). Histopathologically, the severity of DN was classified as grade I (DN I, n = 10; mild mesangial expansion) or grade II (DN II, n = 10; moderate mesangial expansion). We evaluated the expression and localization of PDGF-A, -B, and PDGFR-Α, -Β using in situ hybridization, and quantified PDGF and PDGFR mRNA expression by counting all nuclei, and nuclei surrounded by PDGF-positive cytoplasm and PDGFR-positive cytoplasm, in at least ten randomly selected cross-sections of nonsclerotic glomeruli.ResultsIn all glomeruli, PDGF and PDGFR mRNAs were expressed mainly in glomerular resident cells, predominantly glomerular mesangial and epithelial cells. The percentages of cells positive for PDGF-A and PDGFR-Α mRNA in DN were similar to those in NHK. In contrast, the percentages of PDGF-B and PDGFR-Β mRNA-positive cells in DN were significantly higher than those in NHK, and were significantly higher in DN I than in DN II. The percentages of cells positive for PDGF-B correlated with the PDGFR-Β mRNA level.ConclusionsOur results suggest that the expression of PDGF-B and PDGFR-Β is an important factor in histologically early glomerular lesions of DN.

A Single Nucleotide Polymorphism in KCNQ1 Is Associated With Susceptibility to Diabetic Nephropathy in Japanese Subjects With Type 2 Diabetes

OBJECTIVE Genetic factors have been considered to contribute to the development and progression of diabetic nephropathy. The KCNQ1 gene (potassium voltage-gated channel, KQT-like subfamily, member 1) was originally identified as a strong susceptibility gene for type 2 diabetes in two Japanese genome-wide association studies. In this study, we examined the association of single nucleotide polymorphisms (SNPs) within KCNQ1 with diabetic nephropathy in Japanese subjects with type 2 diabetes. RESEARCH DESIGN AND METHODS We genotyped 33 SNPs in KCNQ1 using 754 type 2 diabetic patients with overt nephropathy and 558 control subjects (an initial study), and we further examined the association of a candidate SNP using three other independent Japanese populations (replications 1–3). RESULTS We found that five SNPs were nominally associated with diabetic nephropathy, and the association of rs2237897 was the strongest. We also found that the T allele frequencies of rs2237897 were consistently higher in the nephropathy groups than in the control groups for all study populations (initial study: 0.33 vs. 0.27; replication 1: 0.32 vs. 0.30; replication 2: 0.33 vs. 0.28; and replication 3: 0.32 vs. 0.28), although the individual associations did not reach statistically significant levels. Combined analysis by a meta-analysis revealed that the T allele of rs2237897 was significantly associated with susceptibility to diabetic nephropathy in Japanese subjects with type 2 diabetes (odds ratio 1.22 [95% CI 1.10–1.34], P = 3.1 × 10–4, corrected P = 0.01). CONCLUSIONS These results suggest that KCNQ1 is a new candidate gene for conferring susceptibility to diabetic nephropathy.

Expression of transcription factor Snai1 and tubulointerstitial fibrosis in progressive nephropathy.

BACKGROUND Tubulointerstitial fibrosis (TIF) is seen as the final stage of progressive nephropathy, and the degree of TIF is reported to be a major determinant in renal outcomes. In recent years, epithelial-mesenchymal transition (EMT) and the zinc-finger transcription factor snail homolog 1 (Snai1) have each been implicated in the mechanism of TIF. The relationship between EMT and these transcription factors is unclear, however, so in this study we attempted to elucidate the correlation between the expression of Snai1 and clinical markers. METHODS We performed immunohistochemical staining on human renal tissue obtained from patients with diabetic nephropathy (DN), IgA nephropathy (IgAN), minimal change disease (MCD) and minor glomerular abnormality (MGA) using anti-Snai1 and anti-vimentin antibodies. We counted Snai1-positive and Snai1/vimentin double positive tubular epithelial cells. RESULTS Snai1 protein was mainly observed in the nuclei of flattened, damaged tubular epithelial cells, especially in IgAN and DN, and positive cell numbers were significantly higher in IgAN than in MGA, MCD or DN. Snai1/vimentin double staining showed that some vimentin-positive tubular epithelial cells also contained Snai1-positive nuclei, and double positive cell numbers were increased in IgAN and DN. Statistical analysis revealed positive correlations between Snai1/vimentin double positive cell numbers and proteinuria and creatinine in IgAN. Positive correlations were also seen between Snai1/vimentin double positive cell numbers and the severity of proteinuria in DN. CONCLUSIONS The results of this study indicate that Snai1 plays an important role in TIF in patients with progressive nephropathy.

Insulin glargine improves glycemic control and quality of life in type 2 diabetic patients on hemodialysis

BACKGROUND Diabetic patients on hemodialysis often experience severe hypoglycemia during intensive insulin therapy using conventional neutral protamine hagedorn (NPH) or nonintensive therapy with premixed insulin. Insulin glargine can simulate normal basal insulin secretion. We investigated the efficacy and safety of switching from NPH to glargine in type 2 diabetes patients on hemodialysis. METHODS Hemodialysis patients who were being treated with NPH-based basal-bolus insulin therapy, regular insulin, NPH insulin or premixed insulin were switched to glargine. The target early morning fasting blood glucose (FBG) level was 110 mg/dL. Any increase in glargine dose was coupled with a reduction in the dose of any regular or rapid-acting insulin analogue as far as possible while maintaining a constant daily insulin dose. FBG, HbA(1c), daily insulin dosage, percentage of basal insulin dose in total daily insulin dose, body weight and incidence of hypoglycemic events were evaluated during the study period. Quality of life (QOL) was measured with a short questionnaire. RESULTS HbA(1c) improved significantly during the observation period after switching. The daily insulin dose was reduced from 20.1 ± 15.2 to 18.1 ± 15.1 U/day, although the change was not statistically significant. FBG decreased significantly from 174.4 ± 58.7 to 126.2 ± 27.7 mg/dL. Body weight measured after dialysis did not change, and there were no changes in hemoglobin or hematocrit. The frequency of hypoglycemic episodes decreased significantly. QOL reports with switching to glargine were improved compared with those before switching. CONCLUSION The results suggest that glargine is useful, can improve QOL of diabetic patients on hemodialysis, and achieve better glycemic control than NPH.

Effects of Multiple Factorial Intervention on Ambulatory BP Profile and Renal Function in Hypertensive Type 2 Diabetic Patients with Overt Nephropathy – A Pilot Study

Abstract Accumulating evidence has shown that diabetic patients are increasing in number, and renal and cardiovascular complications are the most common cause of death in diabetic patients. Thus, it would be of considerable value to identify the mechanisms involved in the progression of renal impairment and cardiovascular injury associated with diabetes. Recent evidence also indicated that multifactorial intervention is able to reduce the risk of cardiovascular disease and death among patients with diabetes and microalbuninuria. In this pilot study, we examined the effects of intensified multifactorial intervention, with tight glucose regulation and the use of valsartan and fluvastatin on ambulatory blood pressure (BP) profile, estimated glomerular filtration rate (eGFR), and urinary albumin to creatinine ratio (UACR), in 20 hypertensive patients (16 male and 4 female) with type 2 diabetes mellitus and overt nephropathy. After 12 months of intensified treatment, office BP, fasting plasma glucose (FPG), and low-density lipoprotein cholesterol (LDLC) were significantly decreased compared to baseline (systolic blood pressure (SBP), 130 ± 2 vs. 150 ± 1 mmHg; diastolic blood pressure (DBP), 76 ± 1 vs. 86 ± 1 mmHg; FPG, 117 ± 5 vs. 153 ± 7 mg/dl; LDLC, 116 ± 8 vs. 162 ± 5 mg/dl, P < 0.0001). Also, compared to the baseline values, the daytime and nighttime ambulatory BP and short-term BP variability were significantly decreased after 12 months. Furthermore, while eGFR was not altered (44.3 ± 5.1 vs. 44.3 ± 6.5 ml/min/1.73 m2, not significant (NS)), UACR showed a significant reduction after 12 months of intensified treatment (1228 ± 355 vs. 2340 ± 381 mg/g-cr, P < 0.05). These results suggest that the intensified multifactorial intervention is able to improve ambulatory BP profile, preserve renal function, and reduce urinary albumin excretion in type 2 diabetic hypertensive patients with overt nephropathy.

Glomerular expression of connective tissue growth factor mRNA in various renal diseases.

SUMMARY:  Connective tissue growth factor (CTGF) is a cysteine‐rich member of a new family of growth regulators. It is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis. The present study was designed to elucidate the role of CTGF in diabetic nephropathy (DN), immunoglobulin A nephropathy (IgA‐N), membranous nephropathy (MN), and minimal change nephrotic syndrome (MCNS). We evaluated the expression and localization of CTGF mRNA in surgically excised renal tissue samples from 10 patients with DN, 10 with IgA‐N, 10 with MN, 10 with MCNS, and 10 normal human kidney (NHK) tissue samples, by using high‐resolution in situ hybridization with digoxigenin‐labelled oligonucleotide. To quantify CTGF mRNA expression, we counted all nuclei, and nuclei surrounded by CTGF‐positive cytoplasm, in at least 10 randomly selected cross‐sections of non‐sclerotic glomeruli, and expressed the results as a percentage of total glomerular cells. In all glomeruli, CTGF mRNA was expressed mainly in glomerular intrinsic cells, including glomerular mesangial and epithelial cells and some cells of Bowmans capsule. The percentage of cells positive for CTGF mRNA was significantly higher in DN and IgA‐N than in MN, MCNS and NHK. However, there was no significant difference in the percentage of CTGF mRNA‐positive cells between DN and IgA‐N. Our study indicates that CTGF may play an important role in the development and progression of glomerulosclerosis in DN and IgA‐N, which are both accompanied by mesangial matrix expansion and comprise two major causes of end‐stage renal failure.

The Influence of a Single Nucleotide Polymorphism within CNDP1 on Susceptibility to Diabetic Nephropathy in Japanese Women with Type 2 Diabetes

Background Several linkage analyses have mapped a susceptibility locus for diabetic nephropathy to chromosome 18q22–23, and polymorphisms within the carnosine dipeptidase 1 gene (CNDP1), located on 18q22.3, have been shown to be associated with diabetic nephropathy in European subjects with type 2 diabetes. However, the association of this locus with diabetic nephropathy has not been evaluated in the Japanese population. In this study, we examined the association of polymorphisms within the CNDP1/CNDP 2 locus with diabetic nephropathy in Japanese subjects with type 2 diabetes. Methodology/Principal Findings We genotyped a leucine repeat polymorphism (D18S880) that is within CNDP1 along with 29 single nucleotide polymorphisms (SNPs) in the CNDP1/CNDP2 locus for 2,740 Japanese subjects with type 2 diabetes (1,205 nephropathy cases with overt nephropathy or with end-stage renal disease [ESRD], and 1,535 controls with normoalbuminuria). The association of each polymorphism with diabetic nephropathy was analysed by performing logistic regression analysis. We did not observe any association between D18S880 and diabetic nephropathy in Japanese subjects with type 2 diabetes. None of the 29 SNPs within the CNDP1/CNDP2 locus were associated with diabetic nephropathy, but a subsequent sex-stratified analysis revealed that 1 SNP in CNDP1 was nominally associated with diabetic nephropathy in women (rs12604675-A; p = 0.005, odds ratio [OR] = 1.76, 95% confidence interval [CI], 1.19−2.61). Rs12604675 was associated with overt proteinuria (p = 0.002, OR = 2.18, 95% CI, 1.32−3.60), but not with ESRD in Japanese women with type 2 diabetes. Conclusions/Significance Rs12604675-A in CNDP1 may confer susceptibility to overt proteinuria in Japanese women with type 2 diabetes.

Expression of connective tissue growth factor mRNA in human IgA nephropathy

AbstractBackground. Connective tissue growth factor (CTGF) is a cysteine-rich member of a new family of growth regulators. Its upregulation is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis. Methods. We evaluated the expression and localization of CTGF mRNA in renal tissues of 20 patients with IgA nephropathy (IgA-N) and 5 normal human kidneys (NHK), using high-resolution in situ hybridization with digoxigenin-labeled oligonucleotide. The expression level of CTGF mRNA was quantitated by counting all nuclei, as well as nuclei surrounded by CTGF mRNA-positive cytoplasm in at least ten randomly selected cross-sections of nonsclerotic glomeruli, and expressing the results as percentage of positive cells. Results. In both IgA-N and NHK, CTGF mRNA was mainly expressed in glomerular intrinsic cells, including mainly glomerular mesangial and epithelial cells, and some endothelial cells and cells of Bowmans capsule. CTGF mRNA-positive cells were abundant in tubulointerstitial fibrotic areas, especially in IgA-N with severe tissue damage. CTGF mRNA expression was also increased in vascular cells in IgA-N. The percentage of cells positive for CTGF mRNA was significantly higher in IgA-N than in NHK. Furthermore, the percentage of cells positive for CTGF mRNA was significantly greater in IgA-N with moderate mesangial proliferative lesions than in IgA-N with mild mesangial proliferative lesions and/or sclerotic lesions. Conclusions. Our study indicates that CTGF may play an important role in the development and progression of glomerulosclerosis and tubulointerstitial fibrosis in IgA-N.