Metabolomic Markers of Kidney Function Decline in Patients With Diabetes: Evidence From the Chronic Renal Insufficiency Cohort (CRIC) Study.

RATIONALE & OBJECTIVE: Biomarkers that provide reliable evidence of future diabetic kidney disease (DKD) are needed to improve disease management. In a cross-sectional study, we previously identified 13 urine metabolites that had levels reduced in DKD compared with healthy controls. We evaluated associations of these 13 metabolites with future DKD progression. STUDY DESIGN: Prospective cohort. SETTING & PARTICIPANTS: 1,001 Chronic Renal Insufficiency Cohort (CRIC) participants with diabetes with estimated glomerular filtration rates (eGFRs) between 20 and 70mL/min/1.73m2 were followed up prospectively for a median of 8 (range, 2-10) years. PREDICTORS: 13 urine metabolites, age, race, sex, smoked more than 100 cigarettes in lifetime, body mass index, hemoglobin A1c level, blood pressure, urinary albumin, and eGFR. OUTCOMES: Annual eGFR slope and time to incident kidney failure with replacement therapy (KFRT; ie, initiation of dialysis or receipt of transplant). ANALYTICAL APPROACH: Several clinical metabolite models were developed for eGFR slope as the outcome using stepwise selection and penalized regression, and further tested on the time-to-KFRT outcome. A best cross-validated (final) prognostic model was selected based on high prediction accuracy for eGFR slope and high concordance statistic for incident KFRT. RESULTS: During follow-up, mean eGFR slope was-1.83±1.92 (SD) mL/min/1.73m2 per year; 359 (36%) participants experienced KFRT. Median time to KFRT was 7.45 years from the time of entry to the CRIC Study. In our final model, after adjusting for clinical variables, levels of metabolites 3-hydroxyisobutyrate (3-HIBA) and 3-methylcrotonyglycine had a significant negative association with eGFR slope, whereas citric and aconitic acid were positively associated. Further, 3-HIBA and aconitic acid levels were associated with higher and lower risk for KFRT, respectively (HRs of 2.34 [95% CI, 1.51-3.62] and 0.70 [95% CI, 0.51-0.95]). LIMITATIONS: Subgroups for whom metabolite signatures may not be optimal, nontargeted metabolomics by flow-injection analysis, and 2-stage modeling approaches. CONCLUSIONS: Urine metabolites may offer insights into DKD progression. If replicated in future studies, aconitic acid and 3-HIBA could identify individuals with diabetes at high risk for GFR decline, potentially leading to improved clinical care and targeted therapies.

Metabolomics in Diabetic Kidney Disease: Unraveling the Biochemistry of a Silent Killer.

The development of new therapies for chronic diseases, such as diabetic kidney disease (DKD), will continue to be hampered by lack of sufficient biomarkers that will provide insights and will be responsive to treatment interventions. The recent application of metabolomic technologies, such as nuclear magnetic resonance and mass spectroscopy, has allowed large-scale analysis of small molecules to be interrogated in a targeted or untargeted manner. Recent advances from both human and animal studies that have arisen from metabolomic analysis have recognized that mitochondrial function and fatty acid oxidation play key roles in the development and progression of DKD. Although many challenges in the technology for clinical chronic kidney disease (CKD) are yet to be validated, there will very likely be ongoing major contributions of metabolomics to develop new biochemical understanding for diabetic and CKD. The clinical application of metabolomics and accompanying bioinformatic tools will likely be a cornerstone of personalized medicine triumphs for CKD.

RNF185 Control of COL3A1 Expression Limits Prostate Cancer Migration and Metastatic Potential.

RNF185 is a RING finger domain-containing ubiquitin ligase implicated in ER-associated degradation. Prostate tumor patient data analysis revealed a negative correlation between RNF185 expression and prostate cancer progression and metastasis. Likewise, several prostate cancer cell lines exhibited greater migration and invasion capabilities in culture upon RNF185 depletion. Subcutaneous inoculation of mouse prostate cancer MPC3 cells stably expressing short hairpin RNA against RNF185 into mice resulted in larger tumors and more frequent lung metastases. RNA-sequencing and Ingenuity Pathway Analysis identified wound-healing and cellular movement among the most significant pathways upregulated in RNF185-depleted lines, compared with control prostate cancer cells. Gene Set Enrichment Analyses performed in samples from patients harboring low RNF185 expression and in RNF185-depleted lines confirmed the deregulation of genes implicated in epithelial-to-mesenchymal transition. Among those, COL3A1 was identified as the primary mediator of RNF185's ability to impact migration phenotypes. Correspondingly, enhanced migration and metastasis of RNF185 knockdown (KD) prostate cancer cells were attenuated upon co-inhibition of COL3A1. Our results identify RNF185 as a gatekeeper of prostate cancer metastasis, partly via its control of COL3A1 availability. IMPLICATIONS: RNF185 is identified as an important regulator of prostate cancer migration and metastasis, in part due to its regulation of COL3A1. Both RNF185 and COL3A1 may serve as novel markers for prostate tumors.

Laser Capture Microscopy RNA Sequencing for Topological Mapping of Synovial Pathology During Rheumatoid Arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune disease in which the joint lining or synovium becomes highly inflamed and majorly contributes to disease progression. Understanding pathogenic processes in RA synovium is critical for identifying therapeutic targets. We performed laser capture microscopy (LCM) followed by RNA sequencing (LCM-RNAseq) to study regional transcriptomes throughout RA synovium. METHODS: Synovial lining, sublining, and vessel samples were captured by LCM from seven patients with RA and seven patients with osteoarthritis (OA). RNAseq was performed on RNA extracted from captured tissue. Principal component analysis was performed on the sample set by disease state. Differential expression analysis was performed between disease states based on log2 fold change and q value parameters. Pathway analysis was performed using the Reactome Pathway Database on differentially expressed genes among disease states. Significantly enriched pathways in each synovial region were selected based on the false discovery rate. RESULTS: RA and OA transcriptomes were distinguishable by principal component analysis. Pairwise comparisons of synovial lining, sublining, and vessel samples between RA and OA revealed substantial differences in transcriptional patterns throughout the synovium. Hierarchical clustering of pathways based on significance revealed a pattern of association between biologic function and synovial topology. Analysis of pathways uniquely enriched in each region revealed distinct phenotypic abnormalities. As examples, RA lining samples were marked by anomalous immune cell signaling, RA sublining samples were marked by aberrant cell cycle, and RA vessel samples were marked by alterations in heme scavenging. CONCLUSION: LCM-RNAseq confirms reported transcriptional differences between the RA synovium and the OA synovium and provides evidence supporting a relationship between synovial topology and molecular anomalies in RA.

RNF185 control of COL3A1 expression limits prostate cancer migration and metastatic potential.

RNF185 is a RING finger domain-containing ubiquitin ligase implicated in ER-associated degradation. Prostate tumor patient data analysis revealed a negative correlation between RNF185 expression and prostate cancer progression and metastasis. Likewise, several prostate cancer cell lines exhibited greater migration and invasion capabilities in culture upon RNF185 depletion. Subcutaneous inoculation of mouse prostate cancer MPC3 cells stably expressing shRNA against RNF185 into mice resulted in larger tumors and more frequent lung metastases. RNA-sequencing and Ingenuity Pathway Analysis identified wound healing and cellular movement among the most significant pathways upregulated in RNF185-depleted, compared to control prostate cancer cells. Gene Set Enrichment Analyses performed in samples from patients harboring low RNF185 expression and in RNF185-depleted lines confirmed the deregulation of genes implicated in EMT. Among those, COL3A1 was identified as the primary mediator of RNF185's ability to impact migration phenotypes. Correspondingly, enhanced migration and metastasis of RNF185 KD prostate cancer cells were attenuated upon co-inhibition of COL3A1. Our results identify RNF185 as a gatekeeper of prostate cancer metastasis, partly via its control of COL3A1 availability.

Systems biology analysis reveals role of MDM2 in diabetic nephropathy.

To derive new insights in diabetic complications, we integrated publicly available human protein-protein interaction (PPI) networks with global metabolic networks using metabolomic data from patients with diabetic nephropathy. We focused on the participating proteins in the network that were computationally predicted to connect the urine metabolites. MDM2 had the highest significant number of PPI connections. As validation, significant downregulation of MDM2 gene expression was found in both glomerular and tubulointerstitial compartments of kidney biopsy tissue from 2 independent cohorts of patients with diabetic nephropathy. In diabetic mice, chemical inhibition of MDM2 with Nutlin-3a led to reduction in the number of podocytes, increased blood urea nitrogen, and increased mortality. Addition of Nutlin-3a decreased WT1+ cells in embryonic kidneys. Both podocyte- and tubule-specific MDM2-knockout mice exhibited severe glomerular and tubular dysfunction, respectively. Interestingly, the only 2 metabolites that were reduced in both podocyte and tubule-specific MDM2-knockout mice were 3-methylcrotonylglycine and uracil, both of which were also reduced in human diabetic kidney disease. Thus, our bioinformatics tool combined with multi-omics studies identified an important functional role for MDM2 in glomeruli and tubules of the diabetic nephropathic kidney and links MDM2 to a reduction in 2 key metabolite biomarkers.