Role of the Immune System in Diabetic Kidney Disease.

PURPOSE OF REVIEW: The purpose of this review is to examine the proposed role of immune modulation in the development and progression of diabetic kidney disease (DKD). RECENT FINDINGS: Diabetic kidney disease has not historically been considered an immune-mediated disease; however, increasing evidence is emerging in support of an immune role in its pathophysiology. Both systemic and local renal inflammation have been associated with DKD. Infiltration of immune cells, predominantly macrophages, into the kidney has been reported in a number of both experimental and clinical studies. In addition, increased levels of circulating pro-inflammatory cytokines have been linked to disease progression. Consequently, a variety of therapeutic strategies involving modulation of the immune response are currently being investigated in diabetic kidney disease. Although no current therapies for DKD are directly based on immune modulation many of the therapies in clinical use have anti-inflammatory effects along with their primary actions. Macrophages emerge as the most likely beneficial immune cell target and compounds which reduce macrophage infiltration to the kidney have shown potential in both animal models and clinical trials.

BMP7-induced-Pten inhibits Akt and prevents renal fibrosis.

Bone morphogenetic protein-7 (BMP-7) counteracts pro-fibrotic effects of TGFß1 in cultured renal cells and protects from fibrosis in acute and chronic renal injury models. Using the unilateral ureteral obstruction (UUO) model of chronic renal fibrosis, we investigated the effect of exogenous-rhBMP-7 on pro-fibrotic signaling pathways mediated by TGFß1 and hypoxia. Mice undergoing UUO were treated with vehicle or rhBMP-7 (300µg/kg i.p.) every other day for eight days and kidneys analysed for markers of fibrosis and SMAD, MAPK, and PI3K signaling. In the kidney, collecting duct and tubular epithelial cells respond to BMP-7 via activation of SMAD1/5/8. Phosphorylation of SMAD1/5/8 was reduced in UUO kidneys from vehicle-treated animals yet maintained in UUO kidneys from BMP-7-treated animals, confirming renal bioactivity of exogenous rhBMP-7. BMP-7 inhibited Collagen Iα1 and Collagen IIIα1 gene expression and Collagen I protein accumulation, while increasing expression of Collagen IVα1 in UUO kidneys. Activation of SMAD2, SMAD3, ERK, p38 and PI3K/Akt signaling occurred during fibrogenesis and BMP-7 significantly attenuated SMAD3 and Akt signaling in vivo. Analysis of renal collecting duct (mIMCD) and tubular epithelial (HK-2) cells stimulated with TGFß1 or hypoxia (1% oxygen) to activate Akt provided further evidence that BMP-7 specifically inhibited PI3K/Akt signaling. PTEN is a negative regulator of PI3K and BMP-7 increased PTEN expression in vivo and in vitro. These data demonstrate an important mechanism by which BMP-7 orchestrates renal protection through Akt inhibition and highlights Akt inhibitors as anti-fibrotic therapeutics.

Gremlin1 plays a key role in kidney development and renal fibrosis.

Gremlin1 (Grem1), an antagonist of bone morphogenetic proteins, plays a key role in embryogenesis. A highly specific temporospatial gradient of Grem1 and bone morphogenetic protein signaling is critical to normal lung, kidney, and limb development. Grem1 levels are increased in renal fibrotic conditions, including acute kidney injury, diabetic nephropathy, chronic allograft nephropathy, and immune glomerulonephritis. We demonstrate that a small number of grem1-/- whole body knockout mice on a mixed genetic background (8%) are viable, with a single, enlarged left kidney and grossly normal histology. The grem1-/- mice displayed mild renal dysfunction at 4 wk, which recovered by 16 wk. Tubular epithelial cell-specific targeted deletion of Grem1 (TEC-grem1-cKO) mice displayed a milder response in the acute injury and recovery phases of the folic acid model. Increases in indexes of kidney damage were smaller in TEC-grem1-cKO than wild-type mice. In the recovery phase of the folic acid model, associated with renal fibrosis, TEC-grem1-cKO mice displayed reduced histological damage and an attenuated fibrotic gene response compared with wild-type controls. Together, these data demonstrate that Grem1 expression in the tubular epithelial compartment plays a significant role in the fibrotic response to renal injury in vivo.

Next Generation RNA Sequencing Analysis Reveals Expression of a Transient EMT Profile During Early Organization of MCF10A Cells in 3D.

RNA sequencing is a technique widely used to identify and characterize gene expression patterns. We demonstrate that this method can be applied to screen expression profiles in mammary epithelial cells cultured in 3D, supported by a natural laminin-rich extracellular matrix, but requires several specific steps in the preparation of the RNA samples. Here we describe the use of RNA sequencing to analyze mRNA patterns in MCF10A human mammary epithelial cells cultured under 3D conditions in a laminin-rich extracellular matrix. We focus on our methods for total RNA extraction at early time points during the formation and maturation of 3D acinus structures in these cultures and provide examples of our results and downstream analysis.

Annexin A8 identifies a subpopulation of transiently quiescent c-kit positive luminal progenitor cells of the ductal mammary epithelium.

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8's association with this breast cancer subgroup we established ANXA8's cellular distribution in the mammary gland and ANXA8's effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (-ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G0/G1 arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8's association with their specific cells of origin.

BMP signalling: agony and antagony in the family.

Bone morphogenetic proteins (BMPs) are secreted extracellular matrix (ECM)-associated proteins that regulate a wide range of developmental processes, including limb and kidney formation. A critical element of BMP regulation is the presence of secreted antagonists that bind and inhibit BMP binding to their cognate Ser/Thr kinase receptors at the plasma membrane. Antagonists such as Noggin, Chordin, Gremlin (Grem1), and twisted gastrulation-1 (Twsg1) have been shown to inhibit BMP action in a range of different cell types and developmental stage-specific contexts. Here we review new developments in the field of BMP and BMP antagonist biology during mammalian development and suggest strategies for targeting these proteins in human disease.

Gremlin1 preferentially binds to bone morphogenetic protein-2 (BMP-2) and BMP-4 over BMP-7.

Gremlin (Grem1) is a member of the DAN family of secreted bone morphogenetic protein (BMP) antagonists. Bone morphogenetic protein-7 (BMP-7) mediates protective effects during renal fibrosis associated with diabetes and other renal diseases. The pathogenic mechanism of Grem1 during diabetic nephropathy (DN) has been suggested to be binding and inhibition of BMP-7. However, the precise interactions between Grem1, BMP-7 and other BMPs have not been accurately defined. In the present study, we show the affinity of Grem1 for BMP-7 is lower than that of BMP-2 and BMP-4, using a combination of surface plasmon resonance and cell culture techniques. Using kidney proximal tubule cells and HEK (human embryonic kidney)-293 cell Smad1/5/8 phosphorylation and BMP-dependent gene expression as readouts, Grem1 consistently demonstrated a higher affinity for BMP-2>BMP-4>BMP-7. Cell-associated Grem1 did not inhibit BMP-2- or BMP-4-mediated signalling, suggesting that Grem1-BMP-2 binding occurred in solution, preventing BMP receptor activation. These data suggest that Grem1 preferentially binds to BMP-2 and this may be the dominant complex in a disease situation where levels of Grem1 and BMPs are elevated.

Targeting cellular drivers and counter-regulators of hyperglycaemia- and transforming growth factor-ß1-associated profibrotic responses in diabetic kidney disease.

Diabetic kidney disease occurs in >30% of patients with type 2 diabetes mellitus and is characterized at source by a maladaptive response in the renal parenchyma to exposure to a glucotoxic-lipotoxic diabetic milieu that courses coincident with hypertension. The consequence of these maladaptive responses is progressive renal injury, which is exacerbated by the development of a chronic inflammatory infiltrate associated with the development of tubulointerstitial fibrosis. The evolution of tubulointerstitial fibrosis is correlated with the loss of functional renal mass and descent towards renal failure. Transforming growth factor-ß1 (TGF-ß1) is a recognized mediator of the profibrotic response of mesangial cells and renal tubular epithelial cells to hyperglycaemia. While euglycaemia remains the goal in the treatment of type 2 diabetes mellitus, the prevention, arrest and reversal of microvascular complications, such as diabetic kidney disease, may be assisted by pharmacological modulation of the effectors of glucotoxicity, such as TGF-ß1. This review focuses on describing how, through reductionist in vitro experimentation focusing on TGF-ß1-related responses to hyperglycaemia, we have identified induced in high glucose-1 (IHG-1), induced in high glucose-2 (IHG-2/Grem1) and the lipoxin-inducible microRNA let-7c as potential targets for harnessing new therapeutic approaches to limit the bioactivity of TGF-ß1 in diabetic kidney disease.

Cadherin juxtamembrane region derived peptides inhibit TGFß1 induced gene expression.

Bioactive peptides in the juxtamembrane regions of proteins are involved in many signaling events. The juxtamembrane regions of cadherins were examined for the identification of bioactive regions. Several peptides spanning the cytoplasmic juxtamembrane regions of E- and N-cadherin were synthesized and assessed for the ability to influence TGFß responses in epithelial cells at the gene expression and protein levels. Peptides from regions closer to the membrane appeared more potent inhibitors of TGFß signaling, blocking Smad3 phosphorylation. Thus inhibiting nuclear translocation of phosphorylated Smad complexes and subsequent transcriptional activation of TGFß signal propagating genes. The peptides demonstrated a peptide-specific potential to inhibit other TGFß superfamily members, such as BMP4.

IHG-1 increases mitochondrial fusion and bioenergetic function.

Induced in high glucose-1 (IHG-1) is a conserved mitochondrial protein associated with diabetic nephropathy (DN) that amplifies profibrotic transforming growth factor (TGF)-ß1 signaling and increases mitochondrial biogenesis. Here we report that inhibition of endogenous IHG-1 expression results in reduced mitochondrial respiratory capacity, ATP production, and mitochondrial fusion. Conversely, overexpression of IHG-1 leads to increased mitochondrial fusion and also protects cells from reactive oxygen species-induced apoptosis. IHG-1 forms complexes with known mediators of mitochondrial fusion-mitofusins (Mfns) 1 and 2-and enhances the GTP-binding capacity of Mfn2, suggesting that IHG-1 acts as a guanine nucleotide exchange factor. IHG-1 must be localized to mitochondria to interact with Mfn1 and Mfn2, and this interaction is necessary for increased IHG-1-mediated mitochondrial fusion. Together, these findings indicate that IHG-1 is a novel regulator of both mitochondrial dynamics and bioenergetic function and contributes to cell survival following oxidant stress. We propose that in diabetic kidney disease increased IHG-1 expression protects cell viability and enhances the actions of TGF-ß, leading to renal proximal tubule dedifferentiation, an important event in the pathogenesis of this devastating condition.

Chromosome 2q31.1 associates with ESRD in women with type 1 diabetes.

Sex and genetic variation influence the risk of developing diabetic nephropathy and ESRD in patients with type 1 diabetes. We performed a genome-wide association study in a cohort of 3652 patients from the Finnish Diabetic Nephropathy (FinnDiane) Study with type 1 diabetes to determine whether sex-specific genetic risk factors for ESRD exist. A common variant, rs4972593 on chromosome 2q31.1, was associated with ESRD in women (P<5×10(-8)) but not in men (P=0.77). This association was replicated in the meta-analysis of three independent type 1 diabetes cohorts (P=0.02) and remained significant for women (P<5×10(-8); odds ratio, 1.81 [95% confidence interval, 1.47 to 2.24]) upon combined meta-analysis of the discovery and replication cohorts. rs4972593 is located between the genes that code for the Sp3 transcription factor, which interacts directly with estrogen receptor α and regulates the expression of genes linked to glomerular function and the pathogenesis of nephropathy, and the CDCA7 transcription factor, which regulates cell proliferation. Further examination revealed potential transcription factor-binding sites within rs4972593 and predicted eight estrogen-responsive elements within 5 kb of this locus. Moreover, we found sex-specific differences in the glomerular expression levels of SP3 (P=0.004). Overall, these results suggest that rs4972593 is a sex-specific genetic variant associated with ESRD in patients with type 1 diabetes and may underlie the sex-specific protection against ESRD.

TGFß and CCN2/CTGF mediate actin related gene expression by differential E2F1/CREB activation.

BACKGROUND: CCN2/CTGF is an established effector of TGFß driven responses in diabetic nephropathy. We have identified an interaction between CCN2 and TGFß leading to altered phenotypic differentiation and inhibited cellular migration. Here we determine the gene expression profile associated with this phenotype and define a transcriptional basis for differential actin related gene expression and cytoskeletal function. RESULTS: From a panel of genes regulated by TGFß and CCN2, we used co-inertia analysis to identify and then experimentally verify a subset of transcription factors, E2F1 and CREB, that regulate an expression fingerprint implicated in altered actin dynamics and cell hypertrophy. Importantly, actin related genes containing E2F1 and CREB binding sites, stratified by expression profile within the dataset. Further analysis of actin and cytoskeletal related genes from patients with diabetic nephropathy suggests recapitulation of this programme during the development of renal disease. The Rho family member Cdc42 was also found uniquely to be activated in cells treated with TGFß and CCN2; Cdc42 interacting genes were differentially regulated in diabetic nephropathy. CONCLUSIONS: TGFß and CCN2 attenuate CREB and augment E2F1 transcriptional activation with the likely effect of altering actin cytoskeletal and cell growth/hypertrophic gene activity with implications for cell dysfunction in diabetic kidney disease. The cytoskeletal regulator Cdc42 may play a role in this signalling response.

Diabetic kidney disease and immune modulation.

Immune modulation is now known to contribute to the development of glomerulosclerosis, tubulointerstitial fibrosis and end-stage renal disease in a large number of kidney diseases. Similarly, diabetic nephropathy is increasingly considered an inflammatory disease, with immune modulation being involved in both the development and progression of the disease. Infiltration of immune cells including macrophages, T cells, B cells and mast cells into the kidney has been reported. A number of pro-inflammatory cytokines and chemokines also play a major role in pathogenesis of diabetic nephropathy. Consequently, a variety of therapeutic strategies involving modulation of the immune response are currently being investigated in diabetic kidney disease.

IHG-1 must be localised to mitochondria to decrease Smad7 expression and amplify TGF-ß1-induced fibrotic responses.

TGF-ß1 is a prototypic profibrotic cytokine and major driver of fibrosis in the kidney and other organs. Induced in high glucose-1 (IHG-1) is a mitochondrial protein which we have recently reported to be associated with renal disease. IHG-1 amplifies responses to TGF-ß1 and regulates mitochondrial biogenesis by stabilising the transcriptional co-activator peroxisome proliferator-activated receptor gamma coactivator-1-alpha. Here we report that the mitochondrial localisation of IHG-1 is pivotal in the amplification of TGF-ß1 signalling. We demonstrate that IHG-1 expression is associated with repression of the endogenous TGF-ß1 inhibitor Smad7. Intriguingly, expression of a non-mitochondrial deletion mutant of IHG-1 (Δmts-IHG-1) repressed TGF-ß1 fibrotic signalling in renal epithelial cells. In cells expressing Δmts-IHG-1 fibrotic responses including CCN2/connective tissue growth factor, fibronectin and jagged-1 expression were reduced following stimulation with TGF-ß1. Δmts-IHG-1 modulation of TGF-ß1 signalling was associated with increased Smad7 protein expression. Δmts-IHG-1 modulated TGF-ß1 activity by increasing Smad7 protein expression as it failed to inhibit TGF-ß1 transcriptional responses when endogenous Smad7 expression was knocked down. These data indicate that mitochondria modulate TGF-ß1 signal transduction and that IHG-1 is a key player in this modulation.

Lipoxins attenuate renal fibrosis by inducing let-7c and suppressing TGFßR1.

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-ß1-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-ß1 suppressed expression of let-7c. In cells pretreated with LXA4, upregulation of let-7c persisted despite subsequent stimulation with TGF-ß1. 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-ß1 signaling pathway, including the TGF-ß receptor type 1. Consistent with this, LXA4-induced upregulation of let-7c inhibited both the expression of TGF-ß receptor type 1 and the response to TGF-ß1. 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-ß1-induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.

The genetics of diabetic nephropathy.

Up to 40% of patients with type 1 and type 2 diabetes will develop diabetic nephropathy (DN), resulting in chronic kidney disease and potential organ failure. There is evidence for a heritable genetic susceptibility to DN, but despite intensive research efforts the causative genes remain elusive. Recently, genome-wide association studies have discovered several novel genetic variants associated with DN. The identification of such variants may potentially allow for early identification of at risk patients. Here we review the current understanding of the key molecular mechanisms and genetic architecture of DN, and discuss the merits of employing an integrative approach to incorporate datasets from multiple sources (genetics, transcriptomics, epigenetic, proteomic) in order to fully elucidate the genetic elements contributing to this serious complication of diabetes.

miR-187 is an independent prognostic factor in breast cancer and confers increased invasive potential in vitro.

PURPOSE: Here, we describe an integrated bioinformatics, functional analysis, and translational pathology approach to identify novel miRNAs involved in breast cancer progression. EXPERIMENTAL DESIGN: Coinertia analysis (CIA) was used to combine a database of predicted miRNA target sites and gene expression data. Using two independent breast cancer cohorts, CIA was combined with correspondence analysis and between group analysis to produce a ranked list of miRNAs associated with disease progression. Ectopic expression studies were carried out in MCF7 cells and miRNA expression evaluated in two additional cohorts of patients with breast cancer by in situ hybridization on tissue microarrays. RESULTS: CIA identified miR-187 as a key miRNA associated with poor outcome in breast cancer. Ectopic expression of miR-187 in breast cancer cells resulted in a more aggressive phenotype. In a test cohort (n = 117), high expression of miR-187 was associated with a trend toward reduced breast cancer-specific survival (BCSS; P = 0.058), and a significant association with reduced BCSS in lymph node-positive patients (P = 0.036). In a validation cohort (n = 470), high miR-187 was significantly associated with reduced BCSS in the entire cohort (P = 0.021) and in lymph node-positive patients (P = 0.012). Multivariate Cox regression analysis revealed that miR-187 is an independent prognostic factor in both cohorts [cohort 1: HR, 7.37; 95% confidence interval (CI), 2.05-26.51; P = 0.002; cohort 2: HR, 2.80; 95% CI, 1.52-5.16; P = 0.001] and in lymph node-positive patients in both cohorts (cohort 1: HR, 13.74; 95% CI, 2.62-72.03; P = 0.002; cohort 2: HR, 2.77; 95% CI, 1.32-5.81; P = 0.007). CONCLUSIONS: miR-187 expression in breast cancer leads to a more aggressive, invasive phenotype and acts as an independent predictor of outcome.

Protein disorder and short conserved motifs in disordered regions are enriched near the cytoplasmic side of single-pass transmembrane proteins.

Intracellular juxtamembrane regions of transmembrane proteins play pivotal roles in cell signalling, mediated by protein-protein interactions. Disordered protein regions, and short conserved motifs within them, are emerging as key determinants of many such interactions. Here, we investigated whether disorder and conserved motifs are enriched in the juxtamembrane area of human single-pass transmembrane proteins. Conserved motifs were defined as short disordered regions that were much more conserved than the adjacent disordered residues. Human single-pass proteins had higher mean disorder in their cytoplasmic segments than their extracellular parts. Some, but not all, of this effect reflected the shorter length of the cytoplasmic tail. A peak of cytoplasmic disorder was seen at around 30 residues from the membrane. We noted a significant increase in the incidence of conserved motifs within the disordered regions at the same location, even after correcting for the extent of disorder. We conclude that elevated disorder within the cytoplasmic tail of many transmembrane proteins is likely to be associated with enrichment for signalling interactions mediated by conserved short motifs.

CCN2/CTGF increases expression of miR-302 microRNAs, which target the TGFß type II receptor with implications for nephropathic cell phenotypes.

Signalling interplay between transforming growth factor-ß (TGFß) and CCN2 [also called connective tissue growth factor (CTGF)] plays a crucial role in the progression of diabetic nephropathy and has been implicated in cellular differentiation. To investigate the potential role of microRNAs (miRNAs) in the mediation of this signalling network, we performed miRNA screening in mesangial cells treated with recombinant human CCN2. Analysis revealed a cohort of 22 miRNAs differentially expressed by twofold or more, including members of the miR-302 family. Target analysis of miRNA to 3'-untranslated regions (3'-UTRs) identified TGFß receptor II (TßRII) as a potential miR-302 target. In mesangial cells, decreased TßRII expression was confirmed in response to CCN2 together with increased expression of miR-302d. TßRII was confirmed as an miR-302 target, and inhibition of miR-302d was sufficient to attenuate the effect of CCN2 on TßRII. Data from the European Renal cDNA Biopsy Bank revealed decreased TßRII in diabetic patients, suggesting pathophysiological significance. In a mouse model of fibrosis (UUO), miR-302d was increased, with decreased TßRII expression and aberrant signalling, suggesting relevance in chronic fibrosis. miR-302d decreased TGFß-induced epithelial mesenchymal transition (EMT) in renal HKC8 epithelial cells and attenuated TGFß-induced mesangial production of fibronectin and thrombospondin. In summary, we demonstrate a new mode of regulation of TGFß by CCN2, and conclude that the miR-302 family has a role in regulating growth factor signalling pathways, with implications for nephropathic cell fate transitions.

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.