High-density single nucleotide polymorphism genome-wide linkage scan for susceptibility genes for diabetic nephropathy in type 1 diabetes: discordant sibpair 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.

Eicosapentaenoic acid ameliorates diabetic nephropathy of type 2 diabetic KKAy/Ta mice: involvement of MCP-1 suppression and decreased ERK1/2 and p38 phosphorylation.

BACKGROUND: Previous studies reported that eicosapentaenoic acid (EPA) was effective against any renal diseases including diabetic nephropathy. Monocyte chemoattractant protein-1 (MCP-1) is a regulating macrophage recruitment protein, which is up-regulated in patients with diabetic nephropathy. The objectives of the present study were to evaluate the effects of EPA including renal MCP-1 expression in diabetic KKAy/Ta mice, MCP-1 production and signal transduction in mouse mesangial cells (MMCs). METHODS: KKAy/Ta mice were injected with EPA ethyl ester (1 g/kg/day) intraperitoneally. Immunohistochemical staining of MCP-1, F4/80, phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phospho-p38 in the renal sections were performed. EPA or specific inhibitors were incorporated in MMCs, and the levels of supernatant MCP-1 were measured. The effect of EPA on ERK1/2, c-jun NH2-terminal kinase (JNK), p38 or phosphoinositide 3-kinase (PI3K) activity in MMCs was examined using Western blot. RESULTS: EPA decreased the levels of serum triglycerides, leptin, urinary albumin and MCP-1, and improved glucose intolerance, mesangial matrix accumulation and tubulointerstitial fibrosis in KKAy/Ta mice. Immunohistochemical staining of MCP-1 and F4/80 in the glomeruli and tubulointerstitial regions was decreased in the EPA-treated group. EPA and specific inhibitors of ERK1/2, JNK and PI3K decreased levels of MCP-1 in MMCs. EPA suppressed phosphorylation of ERK1/2 and p38 in MMCs, and decreased p-ERK positive cells in glomeruli of KKAy/Ta mice. CONCLUSIONS: EPA ameliorates diabetic nephropathy of type 2 diabetic KKAy/Ta mice. We propose that the observed down-regulation of MCP-1 is critically involved in the beneficial effect of EPA, probably in concert with improvement of other clinical parameters.

A case of renal sarcoidosis with complement activation via the lectin pathway.

A 57-year-old woman with pulmonary sarcoidosis was admitted to the hospital because of an elevation of serum creatinine and blood urea nitrogen. On admission, the laboratory data suggested interstitial nephritis without proteinuria and hematuria, whereas a renal biopsy showed granulomatous interstitial nephritis and mild mesangial proliferative glomerulonephritis. Immunoglobulin and C1q deposits were negative, but mannose-binding lectin, C3, C4d, and C5b-9 deposits were marked in the glomerular mesangial areas. The lectin pathway of complement activation may have contributed to the development of glomerular injury in this patient. DNA of Propionibacterium acnes , which is now strongly suspected as the pathogen of sarcoidosis, was detected in the patient's glomerular mesangial cells; tubular epithelial cells, which were involved in granulomatous inflammation; and mononuclear cells in epithelioid granulomas by in situ hybridization. These findings may add new insights to the pathogenesis of renal sarcoidosis, including its relation to infection, because mannose-binding lectin plays a crucial role in the host defense against various pathogens. From this case of renal sarcoidosis, it is hypothesized that P acnes may be involved in pathogenesis of granulomatous interstitial nephritis and that it plays a role in glomerular complement activation via the lectin pathway.

Chromosomal mapping of a quantitative trait locus for the development of albuminuria in diabetic KK/Ta mice.

BACKGROUND: The KK/Ta mouse strain serves as a suitable polygenic model for human type 2 diabetes. We previously reported a genome-wide linkage analysis of KK/Ta alleles contributing to type 2 diabetes and related phenotypes such as fasting hyperglycaemia, glucose intolerance, hyperinsulinaemia, obesity and dyslipidaemia. METHODS: Since KK/Ta mice spontaneously develop renal lesions closely resembling those in human diabetic nephropathy, we investigated the susceptibility loci using the KK/Ta x (BALB/c x KK/Ta) F1 backcross progeny in the present study. RESULTS: A genome-wide analysis of susceptibility loci for albuminuria with microsatellite-based chromosomal maps showed a contributing KK/Ta locus, provisionally designated UA-1, with a significant linkage with the interval on chromosome 2 at 83.0 cM close to the microsatellite marker D2Mit311 with a maximum LOD of 3.5 (chi(2) = 13.2, P = 0.0003). UA-1 was different from the susceptibility loci contributing to type 2 diabetes, which we earlier identified. The mode of inheritance differed from that of hypertension. The progeny homozygous for UA-1 showed significantly higher urinary albumin levels. CONCLUSIONS: Although there were no significant correlations between urinary albumin levels and other diabetic phenotypes, the group of progeny homozygous for both UA-1 and alleles for fasting hyperglycaemia showed the highest urinary albumin levels. Thus, UA-1 appears to increase the risk of diabetic nephropathy, particularly in individuals susceptible to fasting hyperglycaemia, in a gene dosage-dependent manner. There are potentially important candidate genes that may be relevant to diabetic nephropathy.

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.

Effect of pioglitazone on the early stage of type 2 diabetic nephropathy in KK/Ta mice.

Pioglitazone (PIO) has preventive effects on impaired glucose tolerance (IGT) and urinary albumin excretion in diabetes. These effects in the early stage of diabetic nephropathy have not been fully described. Endothelial constitutive nitric oxide synthase (ecNOS) might be one of the mechanisms of glomerular hyperfiltration. The objective of the present study was to evaluate the effect of PIO, including the role of ecNOS on the early stage of diabetic nephropathy in KK/Ta mice. KK/Ta mice were given PIO (10 mg/kg/d) started at 12 or 16 weeks of age for 8 or 4 weeks, respectively. They were divided into 3 groups as follows: early treatment (n = 8), late treatment (n = 8), and control group (n = 12). The urinary albumin/creatinine ratio (ACR), fasting and casual blood glucose levels, ratio of glomerular and Bowman's capsule volume (GB ratio), and systemic blood pressure were measured as phenotypic characterizations. The ecNOS and iNOS protein expression in glomeruli were evaluated by immunofluorescence. PIO, especially early treatment, improved the ACR and the GB ratio, and ecNOS protein expression was decreased in the endothelium of glomerular vessels. The iNOS protein was not detectable. There were no significant changes in the levels of fasting and casual blood glucose and systemic blood pressure among all groups. We conclude that the effect of PIO on microalbuminuria might not be due to changing systemic blood pressure and blood glucose levels. It appears that the decrease of urinary albumin excretion might be related to improvement of glomerular enlargement, including hyperfiltration, since the levels of ecNOS protein were reduced by PIO in the glomerular vessels.

Altered mouse cholinephosphotransferase gene expression in kidneys of type 2 diabetic KK/TA mouse.

It is generally considered that genetic factors may contribute to the susceptibility of type 2 diabetic nephropathy. The purpose of the present study is to identify molecules that contribute to the development and/or progression of this disease. Differential display was performed to isolate genes in the kidney using the KK/Ta mouse model of type 2 diabetes. The differential expression of 8 randomly chosen candidate genes (DN1-8) were verified by reverse-transcriptase polymerase chain reaction (RT-PCR) or Northern blot analysis. DN1-3 (Zn-alpha2-glycoprotein, vascular endothelial growth factor receptor [VEGFR]-2, and lactate dehydrogenase [LDH]) were overexpressed and DN7-8 (peroxisome proliferator-activated receptor [PPAR]-interacting protein [PRIP], unknown) were underexpressed in the KK/Ta mouse kidney. DN4-6 (Ezrin, transcobalamin 2, aldo-ketoreductase) did not differ between KK/Ta and control (BALB/c) mice. DN8 only showed no significant sequence similarity to previously reported genes. Molecular cloning revealed that full-length DN8 shares 89% identity with human cholinephosphotransferase 1 (hCHPT1), and we designated it as "putative" mouse cholinephosphotransferase 1 (mCHPT1). The putative mCHPT1 gene was most closely mapped to the D10Mit94 locus with the highest logarithm of odds (lod) score. In situ hybridization revealed the levels of glomerular putative mCHPT1 in BALB/c mice tended to be slightly higher than those in KK/Ta mice. The altered renal mRNA expression of these genes may be involved in the development and/or progression of diabetic nephropathy.

Gene expression profile in diabetic KK/Ta mice.

BACKGROUND: To identify susceptibility genes for diabetic nephropathy, GeneChip Expression Analysis was employed to survey the gene expression profile of diabetic KK/Ta mouse kidneys. METHODS: Kidneys from three KK/Ta and two BALB/c mice at 20 weeks of age were dissected. Total RNA was extracted and labeled for hybridizing to the Affymetrix Murine Genome U74Av2 array. The gene expression profile was compared between KK/Ta and BALB/c mice using GeneChip expression analysis software. Competitive reverse transcription-polymerase chain reaction (RT-PCR) was used to confirm the results of GeneChip for a selected number of genes. RESULTS: Out of 12,490 probe pairs present on GeneChip, 98 known genes and 31 expressed sequence tags (ESTs) were found to be differentially expressed between KK/Ta and BALB/c kidneys. Twenty-one known genes and seven ESTs that increased in expression and 77 known genes and 24 ESTs that decreased in KK/Ta kidneys were identified. These genes are related to renal function, extracellular matrix expansion and degradation, signal transduction, transcription regulation, ion transport, glucose and lipid metabolism, and protein synthesis and degradation. In the vicinity of UA-1 (quantitative trait locus for the development of albuminuria in KK/Ta mice), candidate genes that showed differential expression were identified, including the Sdc4 gene for syndecan-4, Ahcy gene for S-adenosylhomocysteine hydrolase, Sstr4 gene for somatostatin receptor 4, and MafB gene for Kreisler leucine zipper protein. CONCLUSION: The gene expression profile in KK/Ta kidneys is different from that in age-matched BALB/c kidneys. Altered gene expressions in the vicinity of UA-1 may be responsible for the development of albuminuria in diabetic KK/Ta mice.

Identification of a common risk haplotype for diabetic nephropathy at the protein kinase C-beta1 (PRKCB1) gene locus.

Abnormal activation of protein kinase C-beta isoforms in the diabetic state has been implicated in the development of diabetic nephropathy. It is thus plausible that DNA sequence differences in the protein kinase C-beta1 gene (PRKCB1), which encodes both betaI and betaII isoforms, may influence susceptibility to nephropathy. Nine single-nucleotide polymorphisms (SNP) in PRKCB1 were tested for association with diabetic nephropathy in type I diabetes mellitus, by using both case-control and family-study designs. Allele and genotype distributions of two SNP in the promoter (--1504C/T and --546C/G) differed significantly between case patients and control patients (P < 0.05). These associations were particularly strong with diabetes mellitus duration of <24 yr (P = 0.002). The risk of diabetic nephropathy was higher among carriers of the T allele of the --1504C/T SNP, compared with noncarriers (odds ratio, 2.54; 95% confidence interval, 1.39 to 4.62), and among carriers of the G allele of the --546C/G SNP (odds ratio, 2.45; 95% confidence interval, 1.37 to 4.38). Among individuals with diabetes mellitus duration of >/==" BORDER="0">24 yr, these two SNP were not associated with diabetic nephropathy. These positive findings were confirmed by using the family-based transmission disequilibrium test. The T-G haplotype, with both risk alleles, was transmitted more frequently than expected from heterozygous parents to offspring who developed diabetic nephropathy during the first 24 yr of diabetes mellitus. It is concluded that DNA sequence differences in the promoter of PRKCB1 contribute to diabetic nephropathy susceptibility in type I diabetes mellitus.

Identification of epistatic interaction involved in obesity using the KK/Ta mouse as a Type 2 diabetes model: is Zn-alpha2 glycoprotein-1 a candidate gene for obesity?

The KK/Ta strain serves as a suitable polygenic mouse model for the common form of type 2 diabetes associated with obesity in humans. Recently, we reported the susceptibility loci contributing to type 2 diabetes and related phenotypes in KK/Ta mice. In this study, we focused on expression in the kidneys and liver of KK/Ta and BALB/c mice using differential display (DD) PCR. Zn-alpha(2) glycoprotein-1 (Azgp1) mRNA levels were increased in the kidneys and liver in KK/Ta mice, and sequence analysis revealed a missense mutation. We analyzed the relationship between this polymorphism and various phenotypes in 208 KK/Ta x (BALB/c x KK/Ta) F1 backcross mice. Statistical analysis revealed that Azgp1 and D17Mit218 exhibit a suggestive linkage to body weight (8 weeks) (logarithm of odds 2.3 and 2.9, respectively). Moderate gene-gene interactions were observed at these loci. Adiponectin mRNA levels in 3T3-L1 cells transfected with the expression pcDNA 3.1 vector containing Azgp1 coding sequence of KK/Ta mice were significantly higher than those of BALB/c mice. These results suggest that Azgp1 is a possible candidate gene for regulation of body weight, elucidation of polygenic inheritance, and age-dependent changes in the genetic control of obesity.

Methylenetetrahydrofolate reductase gene polymorphism and susceptibility to diabetic nephropathy in type 1 diabetes.

BACKGROUND: The T allele of the C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with elevated plasma homocysteine levels, and it has been postulated to be a risk factor for the development of diabetic nephropathy. We examined this hypothesis in both a case-control and a follow-up study in individuals with type 1 diabetes. METHODS: In the case-control study, the control group included 310 subjects with normoalbuminuria and diabetes duration of 15 years or greater, and the case group included 88 prevalent cases with end-stage renal disease (ESRD). The follow-up study included 235 subjects with overt proteinuria followed up for 6 years (on average), during which time ESRD developed in 69 subjects. DNA from each individual was genotyped for the C677T MTHFR polymorphism. RESULTS: The frequency of TT homozygotes did not vary significantly among the four groups: 10% in controls, 15% in prevalent cases of ESRD, 13% in cases with new-onset ESRD, and 11% in those who remained proteinuric during follow-up (P = 0.9, 6 df). Similarly, frequency of the T allele varied little among the same groups (range, 33% to 36%; P = 0.9, 3 df) During follow-up, 52 of 323 individuals with diabetic nephropathy died. Total mortality rates were 4.3/100 person-years in TT homozygotes, 2.4/100 person-years in CT heterozygotes, and 3.0/100 person-years in CC homozygotes (P = 0.55, 2 df). CONCLUSION: Using both a large case-control and a follow-up study, we found no evidence that the C677T MTHFR polymorphism has a significant role in the development of diabetic nephropathy in type 1 diabetes.

Effects of candesartan, an angiotensin II type 1 receptor blocker, on diabetic nephropathy in KK/Ta mice.

BACKGROUND: Although therapeutic effects of angiotensin II type 1 receptor blocker (ARB) on renal injury in non-insulin dependant diabetes mellitus (NIDDM) have been demonstrated, the beneficial effects and their mechanisms in diabetic nephropathy have not been well evaluated. METHODS: KK/Ta mice were divided into three groups according to the treatment: candesartan 4 mg/kg/day from 6 to 28 weeks of age (group I; early treatment); from 12 to 28 weeks of age (group II; late treatment); only vehicle (group III). BALB/c mice treated with vehicle were used as controls (group IV). Body weight (BW), systolic blood pressure (SBP), blood glucose, urinary type IV collagen and albumin excretion were measured every 4 weeks. Morphometry and immunohistology of albumin, transforming growth factor-beta1 (TGF-beta1) and Smad7 were performed in all groups. RESULTS: BW and blood glucose were higher in groups I, II and III than in group IV from 8 weeks. SBP was markedly reduced in groups I and II compared with group III (p < 0.05, p < 0.005). Urinary type IV collagen and albumin excretion were increased in group III compared to group IV (p < 0.05, p < 0.005), whereas they were reduced in groups I and II when compared to group III (p < 0.05). Morphometric analysis revealed that the whole glomerular area (WGA), glomerular tuft area (GTA), extracellular matrix area (ECMA) and intraglomerular cell nuclei number (NIGCN) were significantly reduced in groups I, II and IV compared to group III at 28 weeks. In immunohistochemistry, TGF-beta1 expression in both glomeruli and tubules of groups I and II decreased compared to that of group III at 28 weeks, while Smad7 in group III glomeruli was reduced compared to that in groups I and II. CONCLUSIONS: It appears that candesartan reduced urinary type IV collagen and albumin excretion, and attenuated glomerular hypertrophy and mesangial matrix accumulation by the TGF-betaS/Smad signaling pathway in KK/Ta mice with diabetic nephropathy.

Endothelial nitric oxide synthase gene and the development of diabetic nephropathy.

BACKGROUND: Endothelial nitric oxide synthase gene and the development of diabetic nephropathy BACKGROUND: Intron 4 insertion/deletion polymorphism of the constitutive endothelial nitric oxide synthase (ecNOS) gene may be related to diabetic nephropathy. METHODS: A case-control study was performed in three groups of Japanese patients with Type 2 diabetes mellitus, which including 123 patients with advanced diabetic nephropathy, 107 patients with overt proteinuria and normal serum creatinine level, and a control group of 203 patients with normal renal function despite having diabetes for over 10 years. Additionally, logistic regression analysis was used to assess the findings. RESULTS: When we examined the a-deletion/b-insertion in intron 4 of ecNOS gene, the genotype and allele frequencies were not significantly different between the patients with advanced diabetic nephropathy (a/a 2.4, a/b 21.9, b/b 75.5, 'a' 13.4, 'b' 86.6%), the patients with overt proteinuria (a/a 2.8, a/b 15.8, b/b 81.4, 'a' 10.7, 'b' 89.3%) and the control group (a/a 1.4, a/b 21.6, b/b 76.8, 'a' 12.8, 'b' 87.7%). Logistic regression analysis showed that the ecNOS intron4 a-allele frequency was not the related to nephropathy (P = 0.88). CONCLUSION: We conclude that there is no association of the ecNOS gene polymorphism with the development of diabetic nephropathy in Japanese patients with type 2 diabetes.