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

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.

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.

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 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.

Urinary levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), and renal injuries in patients with type 2 diabetic nephropathy.

We examined the correlation among the levels of urinary monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), hyperglycemia, and renal injuries in patients with type 2 diabetic nephropathy. The levels of urinary MCP-1, IL-8, protein excretion, blood urea nitrogen (BUN), serum creatinine (s-Cr), glycohemoglobin A1c (HbA1c), and fasting plasma glucose (FPG) were measured in 24 patients with type 2 diabetic nephropathy and 14 healthy adults as controls. Diabetic nephropathy was classified into three stages: stage 1 = normoalbuminuric, stage 2 = microalbuminuric, and stage 3 = macroalbuminuric. All of the patients showed normal ranges in renal function tests. Levels of urinary MCP-1 in all patients with diabetic nephropathy were significantly higher than those in healthy adults (P < 0.05). The levels of urinary MCP-1 in patients with diabetic nephropathy increased gradually according to the clinical stage of this disease. In contrast, the levels of urinary IL-8 in patients with diabetic nephropathy increased in stages 2 and 3. There was a significant correlation between the levels of urinary IL-8 and those of HbA1c. High glucose may stimulate MCP-1 and/or IL-8 production and their excretion into the urine independently of the phases or pathological lesions of this disease. It appears that IL-8 increased in the early stage of diabetic nephropathy, and MCP-1 increased in the advanced stage of this disease. It was concluded that measurement of urinary MCP-1 and IL-8 may be useful for evaluating the degree of renal injuries in patients with type 2 diabetic nephropathy.