Alexander P. Maxwell

Genome-wide DNA methylation analysis for diabetic nephropathy in type 1 diabetes mellitus

BackgroundDiabetic nephropathy is a serious complication of diabetes mellitus and is associated with considerable morbidity and high mortality. There is increasing evidence to suggest that dysregulation of the epigenome is involved in diabetic nephropathy. We assessed whether epigenetic modification of DNA methylation is associated with diabetic nephropathy in a case-control study of 192 Irish patients with type 1 diabetes mellitus (T1D). Cases had T1D and nephropathy whereas controls had T1D but no evidence of renal disease.MethodsWe performed DNA methylation profiling in bisulphite converted DNA from cases and controls using the recently developed Illumina Infinium® HumanMethylation27 BeadChip, that enables the direct investigation of 27,578 individual cytosines at CpG loci throughout the genome, which are focused on the promoter regions of 14,495 genes.ResultsSingular Value Decomposition (SVD) analysis indicated that significant components of DNA methylation variation correlated with patient age, time to onset of diabetic nephropathy, and sex. Adjusting for confounding factors using multivariate Cox-regression analyses, and with a false discovery rate (FDR) of 0.05, we observed 19 CpG sites that demonstrated correlations with time to development of diabetic nephropathy. Of note, this included one CpG site located 18 bp upstream of the transcription start site of UNC13B, a gene in which the first intronic SNP rs13293564 has recently been reported to be associated with diabetic nephropathy.ConclusionThis high throughput platform was able to successfully interrogate the methylation state of individual cytosines and identified 19 prospective CpG sites associated with risk of diabetic nephropathy. These differences in DNA methylation are worthy of further follow-up in replication studies using larger cohorts of diabetic patients with and without nephropathy.

New susceptibility loci associated with kidney disease in Type 1 diabetes

Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ∼2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2×10−8) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0×10−9). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-β1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1×10−7), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN.

Erythropoietin production in kidney tubular cells

The erythropoietin gene has been cloned in three mammalian species including man and recombinant erythropoietin is now used to treat the anaemia of chronic renal failure. Despite the isolation of the gene the precise cellular location of erythropoietin synthesis remains controversial. We present studies which demonstrate erythropoietin production by kidney tubular cells. Erythropoietin gene expression (messenger RNA) was detected by in situ hybridization using an oligonucleotide gene probe and the translated protein product by immunohistochemistry employing antibodies raised to pure recombinant DNA derived erythropoietin.

A Molecular Variant of Angiotensinogen Is Associated With Diabetic Nephropathy in IDDM

Recent studies have suggested that an inherited predisposition to essential hypertension may increase susceptibility to nephropathy for patients with IDDM. Essential hypertension has been linked to the angiotensinogen (AGT) gene in genetic linkage studies in American and European populations. A molecular variant (M235T), which has a functional effect, has been described with highest plasma AGT levels being associated with the TT genotype. In a case-control study, we have evaluated the role of this functional genetic marker in patients with IDDM and nephropathy and in IDDM patients without nephropathy. We studied 195 IDDM patients, of whom 95 had established diabetic nephropathy; the remaining 100 patients, who had no evidence of microalbuminuria, served as control subjects. All patients were whites born in Northern Ireland. The point mutation in the AGT gene was analyzed using restriction typing. The background frequency of the M235T variant was assessed in 80 healthy blood donors, and the TT genotype was present in 9%. This genotype occurred in 8% of control IDDM patients without nephropathy and 19% of IDDM patients with nephropathy (P = 0.025). The odds ratio for diabetic nephropathy associated with the TT genotype was 2.7 (95% CI 1.04–7.52). There was no relationship between blood pressure and AGT genotypes in the control group. We cannot exclude the possibility that the observed association in the nephropathy group is due to an association between AGT genotype and hypertension. This evidence may help to explain the predisposition to diabetic nephropathy afforded by hypertension and merits further investigation.

Lipoxins Attenuate Renal Fibrosis by Inducing let-7c and Suppressing TGFβR1

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA4) attenuated TGF-β1-induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-β1 suppressed expression of let-7c. In cells pretreated with LXA4, upregulation of let-7c persisted despite subsequent stimulation with TGF-β1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA4 analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-β1 signaling pathway, including the TGF-β receptor type 1. Consistent with this, LXA4-induced upregulation of let-7c inhibited both the expression of TGF-β receptor type 1 and the response to TGF-β1. Overexpression of let-7c mimicked the antifibrotic effects of LXA4 in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA4. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA4-mediated upregulation of let-7c suppresses TGF-β1-induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.

DNA hypermethylation and DNA hypomethylation is present at different loci in chronic kidney disease

Genetic risk factors for chronic kidney disease (CKD) are being identified through international collaborations. By comparison, epigenetic risk factors for CKD have only recently been considered using population-based approaches. DNA methylation is a major epigenetic modification that is associated with complex diseases, so we investigated methylome-wide loci for association with CKD. A total of 485,577 unique features were evaluated in 255 individuals with CKD (cases) and 152 individuals without evidence of renal disease (controls). Following stringent quality control, raw data were quantile normalized and β values calculated to reflect the methylation status at each site. The difference in methylation status was evaluated between cases and controls with resultant P values adjusted for multiple testing. Genes with significantly increased and decreased levels of DNA methylation were considered for biological relevance by functional enrichment analysis using KEGG pathways in Partek Genomics Suite. Twenty-three genes, where more than one CpG per loci was identified with Padjusted < 10−8, demonstrated significant methylation changes associated with CKD and additional support for these associated loci was sought from published literature. Strong biological candidates for CKD that showed statistically significant differential methylation include CUX1, ELMO1, FKBP5, INHBA-AS1, PTPRN2, and PRKAG2 genes; several genes are differentially methylated in kidney tissue and RNA-seq supports a functional role for differential methylation in ELMO1 and PRKAG2 genes. This study reports the largest, most comprehensive, genome-wide quantitative evaluation of DNA methylation for association with CKD. Evidence confirming methylation sites influence development of CKD would stimulate research to identify epigenetic therapies that might be clinically useful for CKD.

The changing pattern of adult primary glomerular disease

BACKGROUND Published biopsy series have shown geographical and temporal variations in the patterns of primary glomerulonephritis (GN). IgA nephropathy is the most common type of GN in most European studies, but there is evidence suggesting that focal segmental glomerulosclerosis (FSGS) is increasingly common in the USA in all ethnic groups. We report the analysis of 30 years of native renal biopsies and the temporal pattern of primary glomerular disease in a single United Kingdom (UK) region. METHODS All 1844 adult native kidney biopsies for 30 years (1976-2005 inclusive) were analysed. The data were divided into three 10-year time frames, and trends in the biopsy rate and diagnosis of primary glomerular disease were considered. RESULTS Biopsy rates increased significantly from 2.02 to 7.08 per hundred thousand population per year (php/year) (chi(2) = 55.9, P < 0.001), and the mean patient age at biopsy rose from 33 to 49 years over the study period (F = 58, P < 0.001). Primary GN was documented in 49% of biopsies; the most common diagnoses within this group were IgA nephropathy (38.8%), membranous nephropathy (29.4%), minimal change disease (9.8%), membranoproliferative GN type 1 (9.6%) and FSGS (5.7%). There was a significant increase in the proportion of IgA nephropathy (chi(2) = 9.6, P = 0.008) and a decrease in membranous nephropathy (chi(2) = 7.2, P = 0.03) over time. The population incidence of FSGS was low and unchanged at 0.18 php/ year from 1986 to 2005. CONCLUSIONS Consistent with several other European studies, IgA nephropathy was the most common primary glomerular disease in this UK region. The diagnosis of FSGS was uncommon with no evidence of a rise in incidence.

Donor ABCB1 Variant Associates with Increased Risk for Kidney Allograft Failure

The impact of variation within genes responsible for the disposition and metabolism of calcineurin inhibitors (CNIs) on clinical outcomes in kidney transplantation is not well understood. Furthermore, the potential influence of donor, rather than recipient, genotypes on clinical endpoints is unknown. Here, we investigated the associations between donor and recipient gene variants with outcome among 4471 white, CNI-treated kidney transplant recipients. We tested for 52 single-nucleotide polymorphisms (SNPs) across five genes: CYP3A4, CYP3A5, ABCB1 (MDR1; encoding P-glycoprotein), NR1I2 (encoding the pregnane X receptor), and PPIA (encoding cyclophilin). In a discovery cohort of 811 patients from Birmingham, United Kingdom, kidney donor CC genotype at C3435T (rs1045642) within ABCB1, a variant known to alter protein expression, was associated with an increased risk for long-term graft failure compared with non-CC genotype (hazard ratio [HR], 1.69; 95% confidence interval [CI], 1.20-2.40; P=0.003). No other donor or recipient SNPs were associated with graft survival or mortality. We validated this association in 675 donors from Belfast, United Kingdom (HR, 1.68; 95% CI, 1.21-2.32; P=0.002), and in 2985 donors from the Collaborative Transplant Study (HR, 1.84; 95% CI, 1.08-3.13; P=0.006). In conclusion, these data suggest that an ABCB1 variant known to alter protein expression represents an attractive candidate for future study and risk stratification in kidney transplantation.

Association Testing of Previously Reported Variants in a Large Case–Control Meta-Analysis of Diabetic Nephropathy

We formed the GEnetics of Nephropathy–an International Effort (GENIE) consortium to examine previously reported genetic associations with diabetic nephropathy (DN) in type 1 diabetes. GENIE consists of 6,366 similarly ascertained participants of European ancestry with type 1 diabetes, with and without DN, from the All Ireland-Warren 3-Genetics of Kidneys in Diabetes U.K. and Republic of Ireland (U.K.-R.O.I.) collection and the Finnish Diabetic Nephropathy Study (FinnDiane), combined with reanalyzed data from the Genetics of Kidneys in Diabetes U.S. Study (U.S. GoKinD). We found little evidence for the association of the EPO promoter polymorphism, rs161740, with the combined phenotype of proliferative retinopathy and end-stage renal disease in U.K.-R.O.I. (odds ratio [OR] 1.14, P = 0.19) or FinnDiane (OR 1.06, P = 0.60). However, a fixed-effects meta-analysis that included the previously reported cohorts retained a genome-wide significant association with that phenotype (OR 1.31, P = 2 × 10−9). An expanded investigation of the ELMO1 locus and genetic regions reported to be associated with DN in the U.S. GoKinD yielded only nominal statistical significance for these loci. Finally, top candidates identified in a recent meta-analysis failed to reach genome-wide significance. In conclusion, we were unable to replicate most of the previously reported genetic associations for DN, and significance for the EPO promoter association was attenuated.

Association of Caveolin-1 Gene Polymorphism With Kidney Transplant Fibrosis and Allograft Failure

CONTEXT Caveolin-1 (CAV1) is an inhibitor of tissue fibrosis. OBJECTIVE To study the association of CAV1 gene variation with kidney transplant outcome, using kidney transplantation as a model of accelerated fibrosis. DESIGN, SETTING, AND PATIENTS Candidate gene association and validation study. Genomic DNA from 785 white kidney transplant donors and their respective recipients (transplantations in Birmingham, England, between 1996 and 2006; median follow-up, 81 months) were analyzed for common variation in CAV1 using a single-nucleotide polymorphism (SNP) tagging approach. Validation of positive findings was sought in an independent kidney transplant donor-recipient cohort (transplantations in Belfast, Northern Ireland, between 1986 and 2005; n = 697; median follow-up, 69 months). Association between genotype and allograft failure was initially assessed by Kaplan-Meier analysis, then in an adjusted Cox model. MAIN OUTCOME MEASURE Death-censored allograft failure, defined as a return to dialysis or retransplantation. RESULTS The presence of donor AA genotype for the CAV1 rs4730751 SNP was associated with increased risk of allograft failure in the Birmingham group (donor AA vs non-AA genotype in adjusted Cox model, hazard ratio [HR], 1.97; 95% confidence interval [CI], 1.29-3.16; P = .002). No other tag SNPs showed a significant association. This finding was validated in the Belfast cohort (in adjusted Cox model, HR, 1.56; 95% CI, 1.07-2.27; P = .02). Overall graft failure rates were as follows: for the Birmingham cohort, donor genotype AA, 22 of 57 (38.6%); genotype CC, 96 of 431 (22.3%); and genotype AC, 66 of 297 (22.2%); and for the Belfast cohort, donor genotype AA, 32 of 48 (67%); genotype CC, 150 of 358 (42%); and genotype AC, 119 of 273 (44%). CONCLUSION Among kidney transplant donors, the CAV1 rs4730751 SNP was significantly associated with allograft failure in 2 independent cohorts.