Chronic inflammation is postulated to be involved in the development of end-stage renal disease in diabetes, but which specific circulating inflammatory proteins contribute to this risk remain ...unknown. To study this, we examined 194 circulating inflammatory proteins in subjects from three independent cohorts with type 1 and type 2 diabetes. In each cohort, we identified an extremely robust kidney risk inflammatory signature (KRIS), consisting of 17 proteins enriched in tumor necrosis factor-receptor superfamily members, that was associated with a 10-year risk of end-stage renal disease. All these proteins had a systemic, non-kidney source. Our prospective study findings provide strong evidence that KRIS proteins contribute to the inflammatory process underlying end-stage renal disease development in both types of diabetes. These proteins point to new therapeutic targets and new prognostic tests to identify subjects at risk of end-stage renal disease, as well as biomarkers to measure responses to treatment of diabetic kidney disease.
We investigated plasma microRNA (miRNA) profiles associated with variation of hyperglycemia, measured as hemoglobin A
(HbA
), in two panels of patients with type 1 diabetes (T1D). Using the HTG ...Molecular Diagnostics EdgeSeq platform, 2,083 miRNAs were measured in plasma from 71 patients included in a screening panel. Quantitative real-time PCR was used to measure the candidate miRNAs in plasma from 95 patients included in an independent replication panel. We found 10 miRNAs replicated in both panels and 4 with high statistical significance. The strongest positive correlations with HbA
were found with miR-125b-5p (
= 0.40,
= 6.0 × 10
) and miR-365a-3p (
= 0.35,
= 5.9 × 10
). The strongest negative correlations were found with miR-5190 (
= -0.30,
= 0.003) and miR-770-5p (
= -0.27,
= 0.008). Pathway analysis revealed that 50 Kyoto Encyclopedia of Genes and Genomes pathways were significantly enriched by genes targeted by these four miRNAs. The axon guidance signaling pathway was enriched (
< 1 × 10
) by genes targeted by all four miRNAs. In addition, three other pathways (Rap1 signaling, focal adhesion, and neurotrophin signaling) were also significantly enriched but with genes targeted by only by three of the identified miRNAs. In conclusion, our study identified four circulating miRNAs that were influenced by variation in hyperglycemia. Dysregulation of these miRNAs, which are associated with hyperglycemia in patients with T1D, may contribute to the development of diabetes complications. However, there are multitudes of possible mechanisms/pathways through which dysregulation of these miRNAs may impact risk of diabetes complications.
Circulating proteins associated with transforming growth factor-β (TGF-β) signaling are implicated in the development of diabetic kidney disease (DKD). It remains to be comprehensively examined which ...of these proteins are involved in the pathogenesis of DKD and its progression to end-stage kidney disease (ESKD) in humans. Using the SOMAscan proteomic platform, we measured concentrations of 25 TGF-β signaling family proteins in four different cohorts composed in total of 754 Caucasian or Pima Indian individuals with type 1 or type 2 diabetes. Of these 25 circulating proteins, we identified neuroblastoma suppressor of tumorigenicity 1 (NBL1, aliases DAN and DAND1), a small secreted protein known to inhibit members of the bone morphogenic protein family, to be most strongly and independently associated with progression to ESKD during 10-year follow-up in all cohorts. The extent of damage to podocytes and other glomerular structures measured morphometrically in 105 research kidney biopsies correlated strongly with circulating NBL1 concentrations. Also, in vitro exposure to NBL1 induced apoptosis in podocytes. In conclusion, circulating NBL1 may be involved in the disease process underlying progression to ESKD, and its concentration in circulation may identify subjects with diabetes at increased risk of progression to ESKD.
Variation in DNA methylation (DNAmet) in white blood cells and other cells/tissues has been implicated in the etiology of progressive diabetic kidney disease (DKD). However, the specific mechanisms ...linking DNAmet variation in blood cells with risk of kidney failure (KF) and utility of measuring blood cell DNAmet in personalized medicine are not clear. We measured blood cell DNAmet in 277 individuals with type 1 diabetes and DKD using Illumina EPIC arrays; 51% of the cohort developed KF during 7 to 20 years of follow-up. Our epigenome-wide analysis identified DNAmet at 17 CpGs (5'-cytosine-phosphate-guanine-3' loci) associated with risk of KF independent of major clinical risk factors. DNAmet at these KF-associated CpGs remained stable over a median period of 4.7 years. Furthermore, DNAmet variations at seven KF-associated CpGs were strongly associated with multiple genetic variants at seven genomic regions, suggesting a strong genetic influence on DNAmet. The effects of DNAmet variations at the KF-associated CpGs on risk of KF were partially mediated by multiple KF-associated circulating proteins and KF-associated circulating miRNAs. A prediction model for risk of KF was developed by adding blood cell DNAmet at eight selected KF-associated CpGs to the clinical model. This updated model significantly improved prediction performance (c-statistic = 0.93) versus the clinical model (c-statistic = 0.85) at
= 6.62 × 10
. In conclusion, our multiomics study provides insights into mechanisms through which variation of DNAmet may affect KF development and shows that blood cell DNAmet at certain CpGs can improve risk prediction for KF in T1D.
Mechanisms underlying the pro gression of diabetic kidney disease to ESKD are not fully understood.
We performed global microRNA (miRNA) analysis on plasma from two cohorts consisting of 375 ...individuals with type 1 and type 2 diabetes with late diabetic kidney disease, and targeted proteomics analysis on plasma from four cohorts consisting of 746 individuals with late and early diabetic kidney disease. We examined structural lesions in kidney biopsy specimens from the 105 individuals with early diabetic kidney disease. Human umbilical vein endothelial cells were used to assess the effects of miRNA mimics or inhibitors on regulation of candidate proteins.
In the late diabetic kidney disease cohorts, we identified 17 circulating miRNAs, represented by four exemplars (miR-1287-5p, miR-197-5p, miR-339-5p, and miR-328-3p), that were strongly associated with 10-year risk of ESKD. These miRNAs targeted proteins in the axon guidance pathway. Circulating levels of six of these proteins-most notably, EFNA4 and EPHA2-were strongly associated with 10-year risk of ESKD in all cohorts. Furthermore, circulating levels of these proteins correlated with severity of structural lesions in kidney biopsy specimens. In contrast, expression levels of genes encoding these proteins had no apparent effects on the lesions. In
experiments, mimics of miR-1287-5p and miR-197-5p and inhibitors of miR-339-5p and miR-328-3p upregulated concentrations of EPHA2 in either cell lysate, supernatant, or both.
This study reveals novel mechanisms involved in progression to ESKD and points to the importance of systemic factors in the development of diabetic kidney disease. Some circulating miRNAs and axon guidance pathway proteins represent potential targets for new therapies to prevent and treat this condition.
This study applies a large proteomics panel to search for new circulating biomarkers associated with progression to kidney failure in individuals with diabetic kidney disease. Four independent ...cohorts encompassing 754 individuals with type 1 and type 2 diabetes and early and late diabetic kidney disease were followed to ascertain progression to kidney failure. During ten years of follow-up, 227 of 754 individuals progressed to kidney failure. Using the SOMAscan proteomics platform, we measured baseline concentration of 1129 circulating proteins. In our previous publications, we analyzed 334 of these proteins that were members of specific candidate pathways involved in diabetic kidney disease and found 35 proteins strongly associated with risk of progression to kidney failure. Here, we examined the remaining 795 proteins using an untargeted approach. Of these remaining proteins, 11 were significantly associated with progression to kidney failure. Biological processes previously reported for these proteins were related to neuron development (DLL1, MATN2, NRX1B, KLK8, RTN4R and ROR1) and were implicated in the development of kidney fibrosis (LAYN, DLL1, MAPK11, MATN2, endostatin, and ROR1) in cellular and animal studies. Specific mechanisms that underlie involvement of these proteins in progression of diabetic kidney disease must be further investigated to assess their value as targets for kidney-protective therapies. Using multivariable LASSO regression analysis, five proteins (LAYN, ESAM, DLL1, MAPK11 and endostatin) were found independently associated with risk of progression to kidney failure. Thus, our study identified proteins that may be considered as new candidate prognostic biomarkers to predict risk of progression to kidney failure in diabetic kidney disease. Furthermore, three of these proteins (DLL1, ESAM, and MAPK11) were selected as candidate biomarkers when all SOMAscan results were evaluated.
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Background: miRNAs are short non-coding RNA molecules involved in gene regulation. Previously, we identified 17 plasma miRNAs (8 risk and 9 protective miRNAs) associated with progression to ESKD in ...diabetes (JASN 2021). Factors that influence the measurement of miRNA concentrations, and thus could impact replication of these findings, including extraction method, miRNA profiling method, and the miRNA source biofluid, have not been investigated.
Methods: Plasma and serum were obtained from patients who participated in the Joslin Kidney Study. Extraction-free plasma and serum miRNA profiles were obtained using the HTG EdgeSeq platform. RNA was also extracted from plasma and used to measure miRNAs by RNA sequencing (RNASeq) and quantitative RT-PCR (qRT-PCR).
Results: Out of 2,083 miRNAs on the EdgeSeq platform, almost all of them (96%) were detected in plasma without RNA extraction, while only 45% of these miRNAs were detected in RNA extracted from the same plasma specimens. The content of miRNAs detected in extracted RNA was significantly enriched for GC sequences in comparison with miRNAs that were lost during RNA extraction. In the specimens with RNA extracted, significantly lower number of miRNAs were detected by RNASeq or qRT-PCR (31% and 26%, respectively) in comparison with the EdgeSeq platform. Regarding the 17 miRNAs associated with ESKD, many of them had different concentrations in serum samples than in plasma and had lower concentration or were not detected in plasma samples with profiled following RNA extraction.
Conclusion: This study demonstrates very strong effects of pre-analytical and analytical factors on concentration of circulating miRNAs. To replicate our findings for 17 circulating miRNAs, it is critical that plasma specimens be used and that miRNAs be measured using the EdgeSeq platform or other platforms that do not require prior extraction of RNA.
Disclosure
E.Satake: None. Z.Md dom: None. H.Kobayashi: Research Support; Bayer Inc. J.Ricca: None. K.O'neil: None. B.Krolewski: None. M.G.Pezzolesi: Research Support; Renalytix. A.Krolewski: None.
Funding
National Institutes of Health (DK131061-01)
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