A metabolomic study of low estimated GFR in non-proteinuric type 2 diabetes mellitus.

AIMS/HYPOTHESIS: We carried out a urinary metabolomic study to gain insight into low estimated GFR (eGFR) in patients with non-proteinuric type 2 diabetes. METHODS: Patients were identified as being non-proteinuric using multiple urinalyses. Cases (n = 44) with low eGFR and controls (n = 46) had eGFR values <60 and ≥60 ml min(-1) 1.73 m(-2), respectively, as calculated using the Modification of Diet in Renal Disease formula. Urine samples were analysed by liquid chromatography/mass spectrometry (LC/MS) and GC/MS. False discovery rates were used to adjust for multiple hypotheses testing, and selection of metabolites that best predicted low eGFR status was achieved using least absolute shrinkage and selection operator logistic regression. RESULTS: Eleven GC/MS metabolites were strongly associated with low eGFR after correction for multiple hypotheses testing (smallest adjusted p value = 2.62 × 10(-14), largest adjusted p value = 3.84 × 10(-2)). In regression analysis, octanol, oxalic acid, phosphoric acid, benzamide, creatinine, 3,5-dimethoxymandelic amide and N-acetylglutamine were selected as the best subset for prediction and allowed excellent classification of low eGFR (AUC = 0.996). In LC/MS, 19 metabolites remained significant after multiple hypotheses testing had been taken into account (smallest adjusted p value = 2.04 × 10(-4), largest adjusted p value = 4.48 × 10(-2)), and several metabolites showed stronger evidence of association relative to the uraemic toxin, indoxyl sulphate (adjusted p value = 3.03 × 10(-2)). The potential effect of confounding on the association between metabolites was excluded. CONCLUSIONS/INTERPRETATION: Our study has yielded substantial new insight into low eGFR and provided a collection of potential urinary biomarkers for its detection.

KCNQ1 SNPS and susceptibility to diabetic nephropathy in East Asians with type 2 diabetes.

AIMS/HYPOTHESIS: A Japanese study had earlier reported that KCNQ1 single-nucleotide polymorphisms (SNPs) may be associated with diabetic nephropathy. To further investigate this finding, we analysed three SNPs, rs2237895, rs2237897 and rs2283228, within the KCNQ1 locus for association with albuminuria among Chinese type 2 diabetic patients residing in Singapore. Albuminuria was analysed as both categorical (micro- and macroalbuminuria) and continuous traits (log(e) albumin/creatinine ratio [ACR]). METHODS: A total of 752 Chinese patients with type 2 diabetes were included in the study. Albuminuria was determined by ACR using spot urine samples, and renal function was approximated using estimated GFR. Genotyping was performed using invader and Taqman assays as appropriate. Multivariate regression analyses were used to analyse the associations between SNPs and renal traits. RESULTS: Significant associations were detected between rs2283228 and macroalbuminuria (p < 0.001, corrected p < 0.01), as well as log(e) ACR (p = 0.004, corrected p = 0.036) after multiple hypothesis testing and adjustment for potential confounding. A trend of increasing OR was observed with increasing severity of diabetic nephropathy (low and high microalbuminuria, macroalbuminuria). rs2237897, previously implicated in the earlier Japanese study, was also associated with macroalbuminuria, but this finding did not remain significant after correction for multiple testing. Meta-analyses of the Chinese and Japanese studies revealed both SNPs to be significantly associated with macroalbuminuria. CONCLUSIONS/INTERPRETATION: Together with the previous Japanese study, our findings support the hypothesis that, in addition to KCNQ1 being an established type 2 diabetes gene, genetic variation in this gene may contribute to susceptibility to diabetic nephropathy in East Asians.

Replication of 13 obesity loci among Singaporean Chinese, Malay and Asian-Indian populations.

OBJECTIVE: Recent genome-wide association studies (GWAS) have identified 38 obesity-associated loci among European populations. However, their contribution to obesity in other ethnicities is largely unknown. METHODS: We utilised five GWAS (N=10 482) from Chinese (three cohorts, including one with type 2 diabetes and another one of children), Malay and Indian ethnic groups from Singapore. Data sets were analysed individually and subsequently in combined meta-analysis for Z-score body-mass index (BMI) associations. RESULTS: Variants at the FTO locus showed the strongest associations with BMI Z-score after meta-analysis (P-values 1.16 × 10(-7)-7.95 × 10(-7)). We further detected associations with nine other index obesity variants close to the MC4R, GNPDA2, TMEM18, QPCTL/GIPR, BDNF, ETV5, MAP2K5/SKOR1, SEC16B and TNKS/MSRA loci (meta-analysis P-values ranging from 3.58 × 10(-4)-1.44 × 10(-2)). Three other single-nucleotide polymorphisms (SNPs) from CADM2, PTBP2 and FAIM2 were associated with BMI (P-value ≤ 0.0418) in at least one dataset. The neurotrophin/TRK pathway (P-value=0.029) was highlighted by pathway-based analysis of loci that had statistically significant associations among Singaporean populations. CONCLUSION: Our data confirm the role of FTO in obesity predisposition among Chinese, Malays and Indians, the three major Asian ethnic groups. We additionally detected associations for 12 obesity-associated SNPs among Singaporeans. Thus, it is likely that Europeans and Asians share some of the genetic predisposition to obesity. Furthermore, the neurotrophin/TRK signalling may have a central role for common obesity among Asians.

Reduced GFR and albuminuria in Chinese type 2 diabetes mellitus patients are both independently associated with activation of the TNF-alpha system.

AIMS/HYPOTHESIS: The involvement of chronic inflammation in albuminuria and renal function was investigated in a cross-sectional study of 320 type 2 diabetic Chinese patients from the Singapore Diabetes Cohort Study. METHODS: Plasma levels of TNF-alpha and its two cellular receptors and of IL-6 and C-reactive protein (CRP) were measured. A composite TNF-alpha score was extracted using principal component analysis. Multiple linear regression analysis was implemented to evaluate the relationship between log( e ) (ln) albumin:creatinine ratio (ACR) and estimated GFR (eGFR) with the inflammatory variables and other clinical covariates. A Bonferroni correction was applied based on the total number of variables entered into regression analyses. RESULTS: ln ACR was significantly associated with TNF-alpha score independently of eGFR even after a Bonferroni correction. TNF-alpha score was also significantly associated with eGFR independently of ln ACR even after correction for multiple testing. These findings were similar when the individual molecules of the TNF-alpha system were analysed separately instead of using the composite TNF-alpha score. No association was observed for IL-6 and CRP with either renal trait. Diabetes duration was a significant predictor for ln ACR but not eGFR. Conversely, age was significantly associated with eGFR but not ln ACR. CONCLUSIONS/INTERPRETATION: Activation of the TNF-alpha system may potentially exert independent effects on ln ACR and eGFR in type 2 diabetes. Because of the study design, one may also consider the possibility that changes in these renal traits may conversely be responsible for such an inflammatory response.

Molecular genetic approaches for studying the etiology of diabetic nephropathy.

A critical challenge faced by clinical nephrologists today is the escalating number of patients developing end stage renal disease, a major proportion of which is attributed to diabetic nephropathy (DN). The need for new measures to prevent and treat this disease cannot be overemphasized. To this end, modern genetic approaches provide powerful tools to investigate the etiology of DN. Human studies have already established the importance of genetic susceptibility for DN. Several major susceptibility loci have been identified using linkage studies. In addition, linkage studies in rodents have pinpointed promising chromosomal segments that influence renal traits. Besides augmenting our understanding of disease pathogenesis, these animal studies may facilitate the cloning of disease susceptibility genes in man through the identification of homologous regions that contribute to renal disease. In human diabetes, various genes have been evaluated for their risk contribution to DN. This widespread strategy has been propelled by our knowledge of the glucose-activated pathways underlying DN. Evidence has emerged that a true association does indeed exist for some candidate genes. Furthermore, the in vivo manipulation of gene expression has shown that these genes can modify features of DN in transgenic and knockout rodent models, thus corroborating the findings from human association studies. Still, the exact molecular mechanisms involving these genes remain to be fully elucidated. This formidable task may be accomplished by continuing to harness the synergy between human and experimental genetic approaches. In this respect, our review provides a first synthesis of the current literature to facilitate this challenging effort.

A genome-wide linkage scan for genes controlling variation in urinary albumin excretion in type II diabetes.

The main hallmark of diabetic nephropathy is elevation in urinary albumin excretion. We performed a genome-wide linkage scan in 63 extended families with multiple members with type II diabetes. Urinary albumin excretion, measured as the albumin-to-creatinine ratio (ACR), was determined in 426 diabetic and 431 nondiabetic relatives who were genotyped for 383 markers. The data were analyzed using variance components linkage analysis. Heritability (h2) of ACR was significant in diabetic (h2=0.23, P=0.0007), and nondiabetic (h2=0.39, P=0.0001) relatives. There was no significant difference in genetic variance of ACR between diabetic and nondiabetic relatives (P=0.16), and the genetic correlation (rG=0.64) for ACR between these two groups was not different from 1 (P=0.12). These results suggested that similar genes contribute to variation in ACR in diabetic and nondiabetic relatives. This hypothesis was supported further by the linkage results. Support for linkage to ACR was suggestive in diabetic relatives and became significant in all relatives for chromosome 22q (logarithm of odds, LOD=3.7) and chromosome 7q (LOD=3.1). When analyses were restricted to 59 Caucasian families, support for linkage in all relatives increased and became significant for 5q (LOD=3.4). In conclusion, genes on chromosomes 22q, 5q and 7q may contribute to variation in urinary albumin excretion in diabetic and nondiabetic individuals.

Angiotensin-I converting enzyme insertion/deletion polymorphism and its association with diabetic nephropathy: a meta-analysis of studies reported between 1994 and 2004 and comprising 14,727 subjects.

AIMS/HYPOTHESIS: The ACE insertion/deletion polymorphism has been examined for association with diabetic nephropathy over the past decade with conflicting results. To clarify this situation, we conducted a comprehensive meta-analysis encompassing all relevant studies that were published between 1994 and 2004 and investigated this potential genetic association. METHODS: A total of 14,727 subjects from 47 studies was included in this meta-analysis. Cases (n=8,663) were type 1 or 2 diabetic subjects with incipient (microalbuminuria) or advanced diabetic nephropathy (proteinuria, chronic renal failure, end-stage renal disease). Control subjects (n=6,064) were predominantly normoalbuminuric. RESULTS: No obvious publication bias was detected. Using a minimal-case definition based on incipient diabetic nephropathy, subjects with the II genotype had a 22% lower risk of diabetic nephropathy than carriers of the D allele (pooled odds ratio [OR]=0.78, 95% CI=0.69-0.88). While there was a reduced risk of diabetic nephropathy associated with the II genotype among Caucasians with either type 1 or type 2 diabetes, the association was most marked among type 2 diabetic Asians (Chinese, Japanese, Koreans) (OR=0.65, 95% CI=0. 51-0.83). This OR is significantly different from the OR of 0.90 (95% CI= 0.78-1.04) that was obtained for type 2 diabetic Caucasians (p=0.019). Using a stricter case definition based on advanced diabetic nephropathy, a comparable risk reduction of 24-32% was observed among the three subgroups, although statistical significance was reached only among Asians. CONCLUSIONS/INTERPRETATION: The results of our meta-analysis support a genetic association of the ACE Ins/Del polymorphism with diabetic nephropathy. These findings may have implications for the management of diabetic nephropathy using ACE inhibitors especially among type 2 diabetic Asians.